Methods for predicting time-to-delivery in pregnant women

ABSTRACT

The present disclosure relates to methods for predicting time-to-delivery in pregnant women. The methods include predicting that a pregnant woman will deliver within a predetermined time frame if PAMG-1 is determined to be present at a level above a predetermined detection threshold in a vaginal fluid sample obtained from the pregnant woman. Also provided are methods for determining a patient&#39;s risk of preterm labor and/or spontaneous rupture of the chorioamniotic membrane.

RELATED APPLICATION

The present application is a divisional and claims the benefit of U.S.patent application Ser. No. 15/885,574, filed Jan. 31, 2018, which is adivisional and claims the benefit of U.S. patent application Ser. No.15/186,140, filed Jun. 17, 2016, now U.S. Pat. No. 9,891,233, issuedFeb. 13, 2018; which claims the benefit of U.S. patent application Ser.No. 14/138,753, filed Dec. 23, 2013, now abandoned, which claims thebenefit of U.S. Provisional Patent Application Ser. Nos. 61/748,310,filed Jan. 2, 2013, and 61/909,238, filed Nov. 26, 2013. The contents ofall of the prior applications are incorporated herein by reference intheir entirety.

FIELD OF THE INVENTION

The present disclosure relates to methods for predictingtime-to-delivery (TTD) in pregnant patients and/or for determining apatient's risk of preterm labor and/or spontaneous rupture of thechorioamniotic membrane.

BACKGROUND OF THE INVENTION

Prediction of time-to-delivery (TTD) is clinically important amongpregnancies at risk for preterm delivery, particularly in regard toadministration of corticosteroids (which have optimal benefit within 24hours to 7 days of administration). In addition, patients at high riskfor preterm birth should deliver in a tertiary care unit. Obstetriciansare tasked with predicting TTD in patients at risk for preterm delivery,particularly given the controversy over the use of repeated steroids.

The American College of Obstetricians and Gynecologists (ACOG) indicatesin its most recent Practice Bulletin on the Management of Preterm Laborthat, while many tests to identify women at risk of preterm birth havebeen proposed and evaluated, only ultrasonography (to determine cervicallength) and fetal fibronectin testing have been shown to have benefit.Ultrasonography or fetal fibronectin testing, or a combination of both,may be useful in identifying women who are at high risk for pretermdelivery. However, their clinical usefulness may rest primarily withtheir ability to identify women who are least likely to deliver (i.e.,the tests' negative predictive value (NPV)), not women who are mostlikely to delivery (i.e., a test with a high positive predictive value(PPV)). Thus, there is an urgent need for a test with a high PPV inorder to accurately predict imminent delivery, and to allow for salutaryintervention.

SUMMARY OF THE INVENTION

As discussed above, what is needed in the art are improved devices andmethods for the accurate diagnosis of patients at risk for imminentdelivery (e.g., within 14 days, 7 days, or 48 hours), particularly inpatients presenting with signs, symptoms or complaints suggestive ofpreterm labor (PTL), but having no clinical evidence of rupture of fetalmembranes (ROM). Such improved devices and methods are of significantvalue to healthcare providers in deciding how to manage their patients,e.g., in determining whether to administer tocolytics in order toprolong gestation, corticosteroids to improve respiratory development ofthe fetus, administration of antibiotics to decrease the risk ofinfection (intra-partum and post-partum), prescription of bed rest,and/or increased observation and fetal monitoring.

Thus, in certain aspects, the present disclosure provides a method ofpredicting time to delivery (TTD), that includes (e.g., comprises,consists essentially of, consists of): (a) contacting a vaginal fluidsample obtained from a pregnant woman with at least two PAMG-1-specificmonoclonal antibodies, wherein at least one of the antibodies binds toPAMG-1 when present in the sample to form a PAMG-1/monoclonal antibodycomplex; (b) detecting the presence of the PAMG-1/monoclonal antibodycomplex in the sample only when the concentration of PAMG-1 in thesample exceeds a predefined detection threshold; and (c) predicting thatthe pregnant woman will deliver within a predetermined time frame ifPAMG-1 is detected. In another aspect, the method of predicting TTDincludes (a) contacting a vaginal fluid sample obtained from a pregnantwoman with at least two PAMG-1-specific monoclonal antibodies, whereinat least one of the antibodies binds to PAMG-1 when present in thesample to form a PAMG-1/monoclonal antibody complex; (b) detecting thepresence of the PAMG-1/monoclonal antibody complex in the sample onlywhen the concentration of PAMG-1 in the sample exceeds a predefineddetection threshold; and (c) predicting that the pregnant woman willdeliver within a predetermined time frame if PAMG-1 is detected; or (d)predicting that the pregnant woman will not deliver within thepredetermined time frame if PAMG-1 is not detected. In some embodiments,step (d) comprises predicting that the pregnant woman will not deliverwithin the predetermined time frame at the time when the vaginal fluidsample was obtained from the pregnant woman. The predetermined timeframe for predicting TTD can be, e.g., within about 48 hours; withinabout 7 days; and/or (iii) within about 14 days. In certain aspects, themethod of predicting TTD has one or more of the following positivepredictive values (PPVs): (i) at least about 39% for predicting TTDwithin 48 hours; (ii) at least about 64% for predicting TTD within 7days; and (iii) at least about 77% for predicting TTD within about 14days. In certain aspects, the method for predicting TTD has one or moreof the following PPVs: (i) about 45.5% for predicting TTD within about48 hours; (ii) about 81.8% for predicting TTD within about 7 days; and(iii) about 90.9% for predicting TTD within 14 days. In some aspects,the method has a negative predictive value (NPV) of greater than about90%. In certain aspects, the method for predicting TTD has one or moreof the following PPVs: (i) about 45.5% for predicting TTD within 48hours; and/or (ii) about 78.3% (e.g., about 78%) for predicting TTDwithin 7 days; and/or (iii) about 87% for predicting TTD within 14 days.In some aspects, the method has a negative predictive value (NPV) of 87%or greater. In still other aspects, the method has one or more of thefollowing NPVs: (i) about 100% for predicting TTD within 48 hours;and/or (ii) about 97.4% (e.g., about 87%) for predicting TTD within 7days; and/or (iii) about 93.6% (e.g., about 84%) for predicting TTDwithin 14 days.

In other aspects, the present disclosure provides a method fordetermining the risk of preterm delivery, wherein the method includes(e.g., comprises, consists essentially of, consists of): (a) contactinga vaginal fluid sample obtained from a pregnant woman with at least twoPAMG-1-specific monoclonal antibodies, wherein at least one of theantibodies binds to PAMG-1 when present in the sample to form aPAMG-1/monoclonal antibody complex; (b) detecting the presence of thePAMG-1/monoclonal antibody complex in the sample only when theconcentration of PAMG-1 in the sample exceeds a predefined detectionthreshold; and (c) predicting that the pregnant woman is at risk ofpreterm delivery if PAMG-1 is detected. In other aspects, the presentdisclosure provides a method for determining the risk of pretermdelivery, wherein the method includes (e.g., comprises, consistsessentially of, consists of): (a) contacting a vaginal fluid sampleobtained from a pregnant woman with at least two PAMG-1-specificmonoclonal antibodies, wherein at least one of the antibodies binds toPAMG-1 when present in the sample to form a PAMG-1/monoclonal antibodycomplex; (b) detecting the presence of the PAMG-1/monoclonal antibodycomplex in the sample only when the concentration of PAMG-1 in thesample exceeds a predefined detection threshold; and (c) predicting thatthe pregnant woman is at risk of preterm delivery if PAMG-1 is detected;or (d) predicting that the pregnant woman is not at risk of pretermdelivery if PAMG-1 is not detected. In some embodiments, step (d)comprises predicting that the pregnant woman is not at risk of pretermdelivery at the time when the vaginal fluid sample was obtained from thepregnant woman.

In still other aspects, the present disclosure provides a method fordetermining a pregnant woman's risk of spontaneous rupture of thechorioamniotic membranes, wherein the method includes (e.g., comprises,consists essentially of, consists of): (a) contacting a vaginal fluidsample obtained from a pregnant woman with at least two PAMG-1-specificmonoclonal antibodies, wherein at least one of the antibodies binds toPAMG-1 when present in the sample to form a PAMG-1/monoclonal antibodycomplex; (b) detecting the presence of the PAMG-1/monoclonal antibodycomplex in the sample only when the concentration of PAMG-1 in thesample exceeds a predefined detection threshold; and (c) determiningthat the pregnant woman is at risk of spontaneous rupture of thechorioamniotic membranes if PAMG-1 is detected. In another aspect, thepresent disclosure provides a method for determining a pregnant woman'srisk of spontaneous rupture of the chorioamniotic membranes, wherein themethod includes: (a) contacting a vaginal fluid sample obtained from apregnant woman with at least two PAMG-1-specific monoclonal antibodies,wherein at least one of the antibodies binds to PAMG-1 when present inthe sample to form a PAMG-1/monoclonal antibody complex; (b) detectingthe presence of the PAMG-1/monoclonal antibody complex in the sampleonly when the concentration of PAMG-1 in the sample exceeds a predefineddetection threshold; and (c) determining that the pregnant woman is atrisk of spontaneous rupture of the chorioamniotic membranes if PAMG-1 isdetected; or (d) determining that the pregnant woman is not at risk ofspontaneous rupture of the chorioamniotic membranes if PAMG-1 is notdetected. In some embodiments, step (d) comprises predicting that thepregnant woman is not at risk of spontaneous rupture of thechorioamniotic membranes at the time when the vaginal fluid sample wasobtained from the pregnant woman.

In certain aspects, the method is for determining the risk ofspontaneous preterm premature rupture of the chorioamniotic membranes.

In another aspect, provided herein is a method for ruling out(predicting as highly unlikely) spontaneous preterm premature ROM orpreterm delivery by a pregnant woman within a predetermined time frame.The method can include: (a) contacting a vaginal fluid sample obtainedfrom a pregnant woman suspected to be at risk for preterm delivery withat least two PAMG-1-specific monoclonal antibodies, wherein at least oneof the antibodies binds to PAMG-1 when present in the sample to form aPAMG-1/monoclonal antibody complex; (b) detecting the presence of anyPAMG-1/monoclonal antibody complex present in the sample only when theconcentration of PAMG-1 in the sample exceeds a predefined detectionthreshold; and (c) ruling out (predicting as highly unlikely)spontaneous preterm premature ROM or preterm delivery within thepredetermined time frame if PAMG-1 is not detected. The predeterminedtime frame can be, e.g., within about 48 hours; within about 7 days;and/or (iii) within about 14 days. In some aspects, the method forruling out (predicting as highly unlikely) spontaneous preterm prematureROM or preterm delivery has a negative predictive value (NPV) of greaterthan about 90%. In some aspects, the method for ruling out (predictingas highly unlikely) spontaneous preterm premature ROM or pretermdelivery has a negative predictive value (NPV) of 87% or greater. Instill other aspects, the method has one or more of the following NPVs:(i) about 100% for ruling out (predicting as highly unlikely)spontaneous preterm premature ROM or preterm delivery within 48 hours;and/or (ii) about 97.4% (e.g., about 87%) for ruling out (predicting ashighly unlikely) spontaneous preterm premature ROM or preterm deliverywithin 7 days; and/or (iii) about 93.6% (e.g., about 84%) for ruling out(predicting as highly unlikely) spontaneous preterm premature ROM orpreterm delivery within 14 days.

In any of the aspects disclosed above, the method can further includedetermining that the fetal membranes of the pregnant woman are intact.The method can also include selecting the pregnant woman for analysis bythe method only if the pregnant woman presents with one or more, two ormore, three or more, or all four of the following: (i) signs, symptomsor complaints suggestive of preterm labor; (ii) a gestational agebetween 20 weeks and 36 weeks, 6 days; (iii) a cervical length of 25 mmor more; and (iv) a cervical dilatation of 3 cm or less. The method canalso include collecting the vaginal fluid sample from the pregnant womanwith a collection device (e.g., a flocked swab). In certain aspects, theflocked vaginal swab provides a 1:4 dilution of any PAMG-1 present inthe vaginal fluid sample. In certain aspects, the flocked swab providesa dilution of any PAMG-1 present in the vaginal fluid sample in a rangeof 1:1 to 1:10. The method can also include any one or more of thefollowing steps: contacting the collection device with a solvent torelease the collected vaginal fluid sample; collecting the vaginal fluidsample over a time period of about 30 seconds; contacting the collectiondevice with the solvent for about 30 seconds after collecting thevaginal fluid sample; contacting the vaginal fluid sample with the atleast two PAMG-1-specific monoclonal antibodies for 5 minutes.

In any of the above aspects, the predetermined detection threshold levelof PAMG-1 can be 4 ng/ml.

In any of the above aspects, the at least two PAMG-1 specific monoclonalantibodies can be used in a lateral flow device. The lateral flow devicecan include a pad region and a test region. The pad region of the testdevice can include one of the at least two PAMG-1 specific monoclonalantibodies and the test region can include the other of the two. Incertain aspects, the PAMG-1 specific monoclonal antibody in the padregion is mobilizable and the PAMG-1 specific monoclonal antibody in thetest region is immobilized. In some aspects, the test region of the testdevice further includes a control region. In some aspects, each of theat least two PAMG-1-specific monoclonal antibodies is an antibodyselected from the group consisting of M271, produced by hybridoma N271,deposited with the Russian National Collection of IndustrialMicroorganisms (VKPM) Depository and assigned accession number VKPM-93;M52, produced by hybridoma N52, deposited with the VKPM and assignedaccession number VKPM-92; and M42, produced by hybridoma N42, depositedwith the VKPM and assigned accession number VKPM-94.

In any of the above aspects, the mobilizable antibody in the pad regioncan be M271, produced by hybridoma N271, deposited with the RussianNational Collection of Industrial Microorganisms (VKPM) Depository andassigned accession number VKPM-93, and the immobilized antibody in thetest region can be M52, produced by hybridoma N52, deposited with theVKPM and assigned accession number VKPM-92.

In certain of the above methods employing a device, the device can bethe device illustrated in FIGS. 1 and 2.

In certain aspects, the present disclosure provides a kit that includes(e.g., comprises, consists essentially of, consists of): (a) a devicefor detecting the presence of PAMG-1 in a vaginal fluid sample whenpresent at a level above a predetermined threshold; and a vaginal swab.In some aspects, the vaginal swab can be flocked. In some aspects, thekit further includes a vial and/or instructions for use. In certainaspects, the predetermined threshold is 4 ng/ml. In yet other aspects,the device includes a first and a second monoclonal antibody specificfor PAMG-1. The first and second PAMG-1-specific monoclonal antibodiescan have different binding specificities and affinities for PAMG-1. Insome aspects, each of the first and second PAMG-1-specific monoclonalantibodies can be an antibody selected from the group consisting ofM271, produced by hybridoma N271, deposited with the Russian NationalCollection of Industrial Microorganisms (VKPM) Depository and assignedaccession number VKPM-93; M52, produced by hybridoma N52, deposited withthe VKPM and assigned accession number VKPM-92; and M42, produced byhybridoma N42, deposited with the VKPM and assigned accession numberVKPM-94. In yet other aspects, the test device is a lateral flow device.The test device can include a pad region and a test region. The padregion of the test device can include one of the first and secondPAMG-1-specific monoclonal antibodies and the test region can includethe other of the first and second PAMG-1-specific monoclonal antibodies.In certain aspects, either or both of the pad and test regions cancontain additional PAMG-1-specific monoclonal antibodies and/or mixturesof the first two PAMG-1-specific monoclonal antibodies. In certainaspects, the PAMG-1-specific monoclonal antibody in the pad region canbe mobilizable and the PAMG-1-specific monoclonal antibody in the testregion can be immobilized. In still other aspects, the mobilizableantibody in the pad region is M271, produced by hybridoma N271,deposited with the Russian National Collection of IndustrialMicroorganisms (VKPM) Depository and assigned accession number VKPM-93,and the immobilized antibody in the test region is M52, produced byhybridoma N52, deposited with the VKPM and assigned accession numberVKPM-92. In some aspects, the test region of the test device furtherincludes a control region. In certain aspects, the kit can be for use ina method of predicting TTD. In other aspects, the kit can be for use ina method of predicting risk of preterm delivery. In still other aspects,the kit can be for use in a method of determining a pregnant woman'srisk of spontaneous rupture of the chorioamniotic membranes (ROM) (e.g.,preterm premature ROM). In some aspects, the device in the kit is thedevice illustrated in FIGS. 1 and 2.

Definitions

As used herein, “time to delivery (TTD),” is the total length of time(e.g., hours, days, weeks) starting from a predetermined beginning timepoint (e.g., the time a patient presents with potential signs of pretermlabor) until a pregnant patient delivers her baby. TTD can be specifiedto be “within a predetermined time frame,” such as, e.g., within about14 days (or within about 7 days, or within about 48 hours) from the timethe prediction is made. As used herein, “predicting TTD” meansdetermining the likelihood of delivery within a predetermined time frame(e.g., within 2, 7, or 14 days. In certain aspects, predicting TTDincludes determining that spontaneous preterm premature ROM or pretermdelivery within the predetermined time point is highly likely. Incertain aspects, predicting TTD includes ruling out spontaneous pretermpremature ROM or preterm delivery within the predetermined time point(i.e., determining spontaneous preterm premature ROM or preterm deliverywithin the predetermined time frame is highly unlikely).

As used herein, “preterm delivery” is defined as delivery before 37weeks gestational age.

As used herein, a pregnant woman who is determined to be “at risk ofpreterm delivery” is one presenting with signs, symptoms, or complaintssuggestive of preterm labor.”

As used herein, a “predefined detection threshold” for a test disclosedherein, is the level (e.g., concentration or amount) at or above which apolypeptide or other substance must be present in a sample (e.g., anundiluted vaginal or cervico-vaginal fluid sample) in order to bedetected (i.e., to give a positive result in a test disclosed herein(e.g., TTD test)).

As used herein, the term “antibody” refers to any polypeptide having abinding affinity for an antigen (e.g., PAMG-1) as specified herein,independent of the method used to obtain the polypeptide. For example,the polypeptide may be a monoclonal antibody or fragment thereof,polyclonal antibody or antigen-binding fragment thereof, or any moleculehaving a binding specificity for a target antigen, as specified herein.

An “antigen” is an entity (e.g., a proteinaceous entity or peptide) towhich a binding molecule specifically binds.

The term “epitope” or “antigenic determinant” refers to a site on anantigen to which a binding molecule (e.g., antibody) specifically binds.

As used herein, an antibody that is “specific for” an antigen (e.g.,PAMG-1) binds to that antigen.

As used herein, a pregnant woman is “suitable for” or “in need of”predicting TTD according to the methods disclosed herein if she meetspredefined criteria, as disclosed herein, such as, but not limited to,having signs, symptoms or complaints suggestive of labor but does nothave clinically detectable rupture of membranes (ROM) (e.g., leakage offluid from cervical os, pooling of fluid in the posterior fornix).

The term “about” as used herein means within an acceptable error rangefor the particular value as determined by one of ordinary skill in theart, which will depend in part on how the value is measured ordetermined, i.e., the limitations of the measurement system. Forexample, “about” can mean within 1 or more than 1 standard deviations,per the practice in the art. Alternatively, “about” can mean a range ofup to 20%, preferably up to 10%, more preferably up to 5%, and morepreferably still up to 1% of a given value. Alternatively, particularlywith respect to biological systems or processes, the term can meanwithin an order of magnitude, preferably within 5-fold, and morepreferably within 2-fold, of a value. Where particular values aredescribed in the application and claims, unless otherwise stated theterm “about” meaning within an acceptable error range for the particularvalue should be assumed.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this disclosure pertains. In case of conflict, thepresent document, including definitions, will control.

All publications, patent applications, patents, and other referencesmentioned herein are incorporated by reference in their entirety. Thematerials, methods, and examples disclosed herein are illustrative onlyand not intended to be limiting.

The details of one or more embodiments of the present disclosure are setforth in the accompanying drawings and the description below. Preferredmethods and materials are described below, although methods andmaterials similar or equivalent to those described herein can also beused in the practice or testing of the present disclosure. Otherfeatures, objects, and advantages of the methods disclosed herein willbe apparent from the description and drawings, and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic longitudinal sectional view and FIG. 2 is a planarview of an exemplary device that can be used to detect the presence ofPAMG-1 in a vaginal fluid sample (e.g., for diagnosing time to delivery(TTD)). The numbers identify the following components of the exemplarydevice: 10—M271 antibody region; 12—pad; 14—test region; 16—controlregion; 18—arrows; 22—nitrocellulose membrane; 24—filter paper membrane;26—adhesive rigid plastic base; 28—partially transparent protective filmwith arrows; and 30—non-transparent protective film.

DETAILED DESCRIPTION OF THE INVENTION

Various aspects of the disclosure are described below.

Overview

The present disclosure provides improved methods for predicting TTDwithin a predetermined time frame (e.g., within about 14 days, 7 days or48 hours). Also provided are methods for determining the risk of pretermdelivery (i.e., delivery before 37 weeks gestational age), and methodsfor determining a pregnant woman's risk of spontaneous rupture ofchorioamniotic membranes (ROM). In general, the methods disclosed hereininclude detecting the presence of PAMG-1 when present at a level above apredefined detection threshold. The presently disclosed methods canpredict TTD and/or rule out spontaneous preterm delivery with a high PPVand a high NPV. A positive test according to the methods disclosedherein can indicate that delivery is imminent (i.e., within about 14days, 7 days, or 48 hours). A negative test (absence of detection ofPAMG-1) indicates that delivery is not likely to occur within 14 days, 7days, or 48 hours. A positive test can also indicate that a pregnantwoman is at risk of spontaneous preterm premature ROM and/or pretermdelivery, while a negative test indicates that a pregnant woman is notat risk of spontaneous preterm premature ROM or preterm delivery. Thus,also provided are methods for ruling out (predicting as highly unlikely)spontaneous preterm premature ROM or preterm delivery.

PAMG-1 is a protein found in high concentrations in amniotic fluid butvery low concentrations in background levels of cervico-vaginaldischarge. In recent years, the medical community has increasinglyaccepted the widespread use of detecting PAMG-1 to aid the provider inconfirming or ruling out rupture of fetal membranes (ROM). The test usedis commercially marketed as the AmniSure® ROM Test, manufactured byAmniSure® International, LLC, Boston, Mass., USA. In a previousinvestigation of the utility of PAMG-1 for the detection of ROM, it wasnoted that in 20 out of the 23 cases where the AmniSure® ROM Test waspositive and standard clinical assessment (i.e., nitrazine, ferning andpooling) was negative, the patient was ultimately determined to havebeen ruptured upon retrospective analysis of her clinical course (see,Lee S E, et al. Obstet Gynecol 2007; 109:634-640). It was later reportedthat for all of the preterm patients in the group that showed signs andsymptoms of labor, delivery followed within 7 days (see, Lee S M, et al.J Matern Fetal Neonatal Med 2009; 22:305-310). The clinical value of apositive AmniSure® ROM Test in the patient presenting with signs andsymptoms of preterm labor (PTL) but without ROM was also investigated.The results demonstrated that the AmniSure® ROM Test was predictive ofdelivery of these patients within 48 hours, 7 days and 14 days (see, LeeM S, et al. J Matern Fetal Neonatal Med. 2012 September; 25(9):1690-8),but the PPV was not optimal.

While not intending to be bound by any one particular theory ormechanism of action, the present methods are believed to providesuperior performance such as, e.g., PPV and NPV, as well as sensitivity(SN) and specificity (SP), at least in part, by providing a diagnostictest that has an increased sensitivity for detecting PAMG-1 in vaginalsecretion samples compared to certain currently available diagnosticmethods. For example, while currently available tests that detect PAMG-1utilize a detection threshold of 5 ng/ml, it is presently discoveredthat adjusting the detection threshold to 4 ng/ml provides asurprisingly improved diagnostic test (e.g., high PPV and high NPV). Itwas unexpected that the 4 ng/ml detection threshold could be used in amethod for predicting TTD with a high PPV, as presently disclosed, sinceit was expected that decreasing the detection threshold below 5 ng/mlwould increase the frequency of false positive results, therebydecreasing the PPV of the test. Moreover, it was not previously realizedthat detecting concentrations of PAMG-1 below 5 ng/ml could be usefulfor predicting TTD, as such small concentrations were thought to be oflittle clinical significance.

It is also presently discovered that the ideal gestational age of apregnant woman suitable for predicting TTD according to the methodsdisclosed herein is specifically between 20 weeks and 36 weeks, 6 days,in order to ensure the highest degree of accuracy of the diagnosticmethod. Also, in certain embodiments, the TTD test disclosed herein hasa high NPV and high PPV, as well as high SN and SP, for the patientpopulation having cervical dilatation of 3 cm or less.

In certain embodiments, a pregnant woman is suitable for and/or selectedfor predicting TTD if she has a gestational age between 20 weeks and 36weeks, 6 days. In certain embodiments, a pregnant woman is suitable forand/or selected for predicting TTD if she has a cervical length of 25 mmor more and/or cervical dilatation of 3 cm or less.

In diagnostic testing, the PPV, or precision rate, is the proportion ofpositive test results that are true positives (such as correctdiagnoses). It is a critical measure of the performance of a diagnosticmethod, as it reflects the probability that a positive test reflects theunderlying condition that is being tested for. Other important measuresinclude negative predictive value (NPV), sensitivity (SN), andspecificity (SP). NPV indicates the proportion of subjects with anegative test result who are correctly identified as not having thecondition being tested. A high NPV for a given test indicates that whenthe test yields a negative result, it is most likely correct in itsassessment, and produces only rarely a false negative result. Thus, forpredicting imminent delivery (e.g., within a specific time frame), ahigh NPV means that the test only rarely predicts that delivery is notimminent when, in reality, it is. The number of true positive resultsand true negative results that a diagnostic test yields (e.g., in aclinical study), can also be combined to determine the sensitivity (SN)and specificity (SP) of a diagnostic test.

PPV can be calculated according to the following formula:

${P\; P\; V} = \frac{{number}\mspace{14mu}{of}\mspace{14mu}{true}\mspace{14mu}{positives}}{{{number}\mspace{14mu}{of}\mspace{14mu}{true}\mspace{14mu}{positives}} + {{number}\mspace{14mu}{of}\mspace{14mu}{false}\mspace{14mu}{positives}}}$

NPV can be calculated according to the following formula:

${N\; P\; V} = \frac{{number}\mspace{14mu}{of}\mspace{14mu}{true}\mspace{14mu}{negatives}}{{{number}\mspace{14mu}{of}\mspace{14mu}{true}\mspace{14mu}{negatives}} + {{number}\mspace{14mu}{of}\mspace{14mu}{false}\mspace{14mu}{negatives}}}$

SN can be calculated according to the following formula:

${S\; N} = \frac{{number}\mspace{14mu}{of}\mspace{14mu}{true}\mspace{14mu}{positives}}{{{number}\mspace{14mu}{of}\mspace{14mu}{true}\mspace{14mu}{positives}} + {{number}\mspace{14mu}{of}\mspace{14mu}{false}\mspace{14mu}{negatives}}}$

SP can be calculated according to the following formula:

${S\; P} = \frac{{number}\mspace{14mu}{of}\mspace{14mu}{true}\mspace{14mu}{negatives}}{{{number}\mspace{14mu}{of}\mspace{14mu}{true}\mspace{14mu}{negatives}} + {{number}\mspace{14mu}{of}\mspace{14mu}{false}\mspace{14mu}{positives}}}$

In certain aspects, the methods disclosed herein have a PPV of at leastabout 77% for predicting TTD within 14 days. In a specific embodiment,the PPV for predicting TTD within about 14 days is about 90.9% (e.g.,about 91%). In another specific embodiment, the PPV for predicting TTDwithin about 14 days is about 87%. It is to be appreciated that thepresent methods also encompass methods for predicting TTD within about14 days that have a PPV of at least about, e.g., 75%, 76%, 78%, 79%,80%, 81%, 82%, 83%, 84% 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99% or 100%. In certain aspects, the PPV forpredicting TTD within about 14 days is in the range of about 70%-100%,about 80-%-100%, about 85%-100%, or about 90%-100%.

The methods disclosed herein have a PPV of at least about 64% forpredicting TTD within about 7 days. In a specific embodiment, the PPVfor predicting TTD within about 7 days is 81.8%. In another specificembodiment, the PPV for predicting TTD within about 7 days is about78.3% (e.g., about 78%). It is to be appreciated that the presentmethods also encompass methods for predicting TTD within about 7 daysthat have a PPV of at least about, e.g., 65%, 66%, 67%, 68%, 69%, 70%,71%, 72%, 73%, 74%, 75%, 76%, 78%, 79%, 80%, 81%, 82%, 83%, 84% 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or100%. In certain aspects, the PPV for predicting TTD within 7 days is inthe range of about 60%-100%, about 65%-100%, about 70%-100%, about75%-100%, or about 80%-100%.

In other aspects, the methods disclosed herein have a PPV of forpredicting TTD within about 48 hours of at least about 39%. In aspecific embodiment, the PPV for predicting TTD within about 48 hours is45.5%. It is to be appreciated that the present methods also encompassmethods for predicting TTD within about 48 hours that have a PPV of atleast about, e.g., 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%,50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%,64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%,78%, 79%, 80%, 81%, 82%, 83%, 84% 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%. In certain aspects, thePPV for predicting TTD within about 48 hours using the present methodsis in the range of about 39%-100%, about 40%-100%, or about 45-100%.

In certain aspects, the NPV for predicting TTD according to the methoddisclosed herein is at least about 90% (e.g., about 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99% or 100%) for predicting TTD within about 48hours, 7 days or 14 days.

In certain aspects, the SN for predicting TTD according to the methoddisclosed herein is at least about 70% (e.g., at least about 70%, 71%,72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84% 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or100%) for predicting TTD within about 48 hours, 7 days or 14 days.

In certain aspects, the SP for predicting TTD according to the methoddisclosed herein is at least about 70% (e.g., at least about 70%, 71%,72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84% 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or100%) for predicting TTD within about 48 hours, 7 days or 14 days.

In certain embodiments, the present methods provide a diagnostic testthat has a PPV of about 90.9%, and/or a NPV of about 93.6%, and/or an SNof about 80%, and/or a SP of about 97.3% for predicting TTD within about14 days. In certain embodiments, the present methods provide adiagnostic test that has a PPV of about 91%, and/or a NPV of about 94%,and/or an SN of about 80%, and/or a SP of about 97.3% for predicting TTDwithin about 14 days.

In certain embodiments, the present methods provide a diagnostic testthat has a PPV of about 87%, a NPV of about 93.6%, an SN of about 80%,and a SP of about 96.1% for predicting TTD within about 14 days. Incertain embodiments, the present methods provide a diagnostic test thathas a PPV of about 87%, and/or a NPV of about 94%, and/or an SN of about80%, and/or a SP of about 96% for predicting TTD within about 14 days.

In certain embodiments, the present methods provide a diagnostic testthat has a PPV of 81.8%, a NPV of 97.4%, a SN of 90%, and a SP of 95%,for predicting TTD within about 7 days.

In certain embodiments, the present methods provide a diagnostic testthat has a PPV of about 78.3%, and/or a NPV of about 97.4%, and/or a SNof about 90%, and/or a SP of about 93.8%, for predicting TTD withinabout 7 days. In certain embodiments, the present methods provide adiagnostic test that has a PPV of about 78%, and/or a NPV of about 97%,and/or a SN of about 90%, and/or a SP of about 94%, for predicting TTDwithin about 7 days.

In certain embodiments, the present methods provide a diagnostic testthat has a PPV of about 45.5%, a NPV of about 100%, a SN of about 100%,and a SP of about 86.7%, for predicting TTD within about 48 hours.

It is to be understood that the above values and ranges can be adjustedto include confidence intervals (e.g., 95% confidence intervals), asshown, e.g., in Example 2, Tables 2 and 4, below.

The methods disclosed herein are also useful for determining a patient's(i.e., pregnant woman's) risk of preterm delivery. Preterm delivery isdefined herein as delivery before 37 weeks gestational age. A positivetest obtained according to the methods disclosed herein (i.e., detectionof PAMG-1 at a level at or above a predefined detection threshold in avaginal fluid sample) indicates that a patient is at risk of pretermdelivery. In certain embodiments, a patient is selected for testing forrisk of preterm delivery if the patient presents with one or more of thefollowing signs: (i) a gestational age between 20 weeks and 36 weeks, 6days; and/or (ii) a cervical length of 25 mm or more; and/or (iii) acervical dilatation of 3 cm or less.

The methods disclosed herein are also useful for determining a pregnantwoman's risk of spontaneous rupture of the chorioamniotic membranes(ROM), such as, e.g., preterm premature ROM. Spontaneous ROM typicallyoccurs as part of the normal labor process. However, preterm prematureROM (e.g., before reaching 37 weeks gestational age) can also occur. Itis advantageous to be able to determine a patient's risk of spontaneousROM, including preterm premature ROM, so that appropriate interveningmeasures (e.g., administration of tocolytics in order to prolonggestation, corticosteroids to improve respiratory development of thefetus, administration of antibiotics to decrease the risk of infection(intra-partum and post-partum), prescription of bed rest, and/orincreased observation and fetal monitoring) can be taken, if necessary.In certain embodiments, a patient is selected for testing for risk ofspontaneous ROM if the patient presents with one or more of thefollowing signs: (i) a gestational age between 20 weeks and 36 weeks, 6days; and/or (ii) a cervical length of 25 mm or more; and/or (iii) acervical dilatation of 3 cm or less.

PAMG-1

PAMG-1 was isolated in 1977 from amniotic fluid by D. Petrunin and wasoriginally referred to as specific alpha-1 globulin of placenta (D.Petrunin, et al., “Immunological Identification of Organ Specificalpha-1 Globulin of Human Placenta and Its Content in the AmnioticFluid,” in Akusherstvo i Ginekologiya, 1977, N 1, pp. 64-65, Moscow,USSR).

The exemplary steps of the isolation of PAMG-1 from amniotic fluid ofpregnant women are outlined in Table 1, and discussed, below. It is tobe understood, however, that PAMG-1 can be isolated according to anysuitable method known in the art, from any suitable source.

TABLE 1 Exemplary Steps of Isolation of PAMG-1 Purity Yield Steps ofIsolation (%) (%) Amniotic fluid 16-25 weeks pregnancy 4 100Precipitation by 0.5% lanthanum chloride 25 90 Precipitation by ammoniumsulphate at 50% saturation 35 70 Precipitation by lithium sulphate at60% saturation 60 60 Reverse Phase Chromatography Separation 90 30

PAMG-1 was isolated from the amniotic fluid of women at 16 to 25 weeksof gestation. The fluid was obtained from women whose pregnancy wasterminated due to medical considerations. A 10% solution of lanthanumchloride was added at the volumetric ratio 20:1 (so that its finalconcentration was 0.5%) to the amniotic fluid and kept at 4° C. for 18hours. The precipitate was further separated by centrifugation at 8,000rpm for 30 minutes. The precipitate was dissolved in a saturatedsolution of Na₂HPO₄ and then the precipitate of insoluble lanthanumsalts (produced in the process of centrifugation at 8,000 rpm for 30minutes) was separated. The resulting solution was fractionated with 50%saturated ammonium sulphate by incubating at 4° C. for 18 hours, and theresulting precipitate was dissolved in distilled water in such a way asto restore the volume of the dissolved precipitation fractions to theinitial volume of the amniotic fluid. Then, the solution wasprecipitated by 60% saturated lithium sulphate, and the precipitate wasdissolved in a small amount of distilled water. After dialysis, theadmixtures were adsorbed with calcium pyrophosphate by adding an equalvolume of moisture absorbent to the protein solution, intermixing andincubating for 10-15 minutes, and separating the absorbent bycentrifugation.

The molecular weight of PAMG-1 was first reported as 32 kDa(Boltovskaya, M. N. et al., “Histochemical and Clinico-Diagnostic Studyof the Placental Alpha-Microglobulin [PAMG-1] Using MonoclonalAntibodies,” in Bulletin of Experimental. Biology and Medicine, 1991,No. 10, pp. 397-400); however, it is generally accepted now that PAMG-1has a molecular weight of 34 kDa (see, e.g., Pollet-Villard et al. (AmerJ Perinatol 2011 June; 28(6):489-94)). PAMG-1 is a protein that ispresent in the serum, amniotic fluid and vaginal secretion of pregnantwomen. PAMG-1 exists in amniotic fluid at a concentration about at least100 times greater than in the serum of pregnant women and at least 3000times greater than in vaginal secretions of pregnant women in theabsence of fetal membranes rupture. As a result, even when a smallamount of amniotic liquid (about 1/100 of one drop per 1 ml of vaginalsecretion) is dissolved in a vaginal secretion sample, a sufficientamount of PAMG-1 is present in this vaginal secretion sample to indicatethat fetal membrane rupture has taken place. Further, because of the lowconcentration of PAMG-1 in blood serum, the insignificant admixture ofblood serum to the vaginal fluid sample (10-15%) does not affect theresults produced by the devices and methods of the present disclosure.Detection of PAMG-1 for the diagnosis of ROM has been shown to besuperior to the detection of other amniotic proteins such as, e.g.,IGFBP-1, a 28 kDa protein (see, Pollet-Villard et al. (supra) andEuropean Guidelines on preterm labor (The Journal of Maternal-Fetal andNeonatal Medicine, 2011; Early Online, 1-9)).

Because the presence of amniotic fluid in a vaginal secretion can beindicative of a fetal membrane rupture, the detection of the amnioticprotein PAMG-1 in vaginal secretion can be used to detect fetal membranerupture. However, it is presently discovered that the methods disclosedherein can be used to detect PAMG-1 in vaginal secretions even in theabsence of detectable ROM to accurately predict TTD, by adjusting thedetection threshold of PAMG-1 to about 4 ng/ml. While not intending tobe bound by theory or limited to any one particular mechanism of action,it is believed that PAMG-1 is transudated through chorioamniotic poresin fetal membranes during uterine contractions that occur when deliveryis imminent (i.e., will occur within, e.g., 14 days, 7 days, or 48hours). Degradation of extracellular matrix of fetal membranes due toinflammatory process of labor and or infection may also lead to thefinding of increased levels of PAMG-1 in cervico-vaginal secretions.

PAMG-1 Antibodies

The methods disclosed herein encompass detecting the presence of PAMG-1protein in vaginal secretion samples obtained from pregnant women.PAMG-1 protein can be detected according to any suitable method known inthe art.

An exemplary method for the detection of PAMG-1 in vaginal fluid samplesincludes, but is not limited to, immunoassay (e.g., ELISA), using, e.g.,PAMG-1 specific antibodies (e.g., monoclonal antibodies orantigen-binding fragments thereof) described herein.

PAMG-1 antibodies, as disclosed herein, can detect very lowconcentrations of PAMG-1. For example, concentration of 0.05 ng/mlPAMG-1 can be detected. Because the maximum concentration of PAMG-1 inserum is about 25 ng/ml, as compared to a minimum concentration of about1680 ng/ml in amniotic fluid, and because the background concentrationof PAMG-1 in vaginal secretions is very low, about 0.2 ng/ml, a lowerthreshold level for PAMG-1 can be used in the methods of the presentdisclosure for detecting the occurrence of amniotic fluid in the vagina.It is presently discovered that a predefined threshold of about 4 ng/mlfor PAMG-1 can be used in the methods disclosed herein.

As a result, the devices and methods of the present disclosure are notinfluenced by the presence of vaginitis or other variables, which had anegative impact on the accuracy of prior methods for detecting fetalmembrane ruptures. The maximum concentration of PAMG-1 in inflammationexudate is 3 ng/ml (see, e.g., U.S. Pat. No. 7,709,272 by Fuks et al.).The same concentration of PAMG-1 may occur if blood serum admixture tovaginal secretion does not exceed 10-15%. In addition, a large ratio ofconcentrations serum-to-amniotic PAMG-1 makes the devices and methods ofthe present disclosure significantly less likely to produce falsepositive results due to the presence of blood serum in vaginalsecretions, even with a low PAMG-1-detection threshold.

The present disclosure provides methods and devices for predicting TTDat a PAMG-1 detection threshold of about 4 ng/ml. The detectionthreshold can be adjusted by selecting PAMG-1 antibodies (e.g., a pairof PAMG-1 binding antibodies) with specific binding affinities forPAMG-1, such that the combination of the PAMG-1 binding antibodiesprovides the desired detection threshold. The detection threshold canalso be adjusted, e.g., by using at least one or more additionalantibodies in test region (e.g., test region 14 in FIGS. 1 and 2)against PAMG-1 to adjust the predefined detection threshold (see U.S.Pat. No. 7,709,272 by Fuks et al.), or by adjusting the test procedure,as discussed in detail below.

PAMG-1 polypeptide separated from body fluids, produced recombinantly,or by chemical synthesis, and fragments or other derivatives or analogsthereof, including fusion proteins, may be used as an immunogen togenerate antibodies that recognize the PAMG-1 polypeptide. Theantibodies disclosed herein can include an immunoglobulin heavy chain ofany isotype (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1,IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass of immunoglobulin molecule.PAMG-1 antibodies may have both a heavy and a light chain.

Antibodies (including full length antibodies), monoclonal antibodies(including full length monoclonal antibodies), polyclonal antibodies,multispecific antibodies (e.g., bispecific antibodies), human, humanizedor chimeric antibodies, and antibody fragments, e.g., Fab fragments,F(ab′) fragments, fragments produced by a Fab expression library,epitope-binding fragments of any of the above, and engineered forms ofantibodies, e.g., scFv molecules, so long as they exhibit the desiredactivity, e.g., binding to PAMG-1, can be used to perform the methodsdisclosed herein. Anti-PAMG-1 antibodies as, e.g., disclosed herein, mayrecognize PAMG-1 from one or more different mammalian species.Alternatively, an antibody disclosed herein may be specific for a singleform of PAMG-1. In certain embodiments, an anti-PAMG-1 antibody isspecific for human PAMG-1.

Epitopes can be formed both from contiguous amino acids or noncontiguousamino acids juxtaposed by tertiary folding of a protein. Epitopes formedfrom contiguous amino acids are typically retained on exposure todenaturing solvents whereas epitopes formed by tertiary folding aretypically lost on treatment with denaturing solvents. An epitopetypically includes at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or15 amino acids in a unique spatial conformation. Methods of determiningspatial conformation of epitopes include, for example, X-raycrystallography and 2-dimensional nuclear magnetic resonance. See, e.g.,Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, G.E. Morris, Ed. (1996).

Antibodies that recognize the same or overlapping epitopes can beidentified in a simple immunoassay showing the ability of one antibodyto block the binding of another antibody to a target antigen, i.e., acompetitive binding assay. Competitive binding is determined in an assayin which the binding molecule being tested inhibits specific binding ofa reference binding molecule to a common antigen, such as PAMG-1.Numerous types of competitive binding assays are known, for example:solid phase direct or indirect radioimmunoassay (RIA); solid phasedirect or indirect enzyme immunoassay (EIA) sandwich competition assay(see Stahli et al., Methods in Enzymology 9:242 (1983)); solid phasedirect biotin-avidin EIA (see Kirkland et al., J. Immunol. 137:3614(1986)); solid phase direct labeled assay, solid phase direct labeledsandwich assay (see Harlow and Lane, Antibodies: A Laboratory Manual,Cold Spring Harbor Press (1988)); solid phase direct label RIA using1-125 label (see Morel et al., Mol. Immunol. 25(1):7 (1988)); solidphase direct biotin-avidin EIA (Cheung et al., Virology 176:546 (1990));and direct labeled RIA. (Moldenhauer et al., Scand. J. Immunol. 32:77(1990)).

Typically, such an assay involves the use of purified antigen bound to asolid surface or cells bearing either of these, an unlabeled testbinding molecule and a labeled reference binding molecule. Competitiveinhibition is measured by determining the amount of label bound to thesolid surface or cells in the presence of the test binding molecule.Usually the test binding molecule is present in excess. Usually, when acompeting binding molecule is present in excess, it will inhibitspecific binding of a reference binding molecule to a common antigen byat least 50-55%, 55-60%, 60-65%, 65-70% 70-75% or more.

Various procedures known in the art may be used for the production ofpolyclonal antibodies to PAMG-1 polypeptide or derivative or analogthereof. For the production of antibody, various host animals can beimmunized by injection with the PAMG-1 polypeptide, or a derivative(e.g., fragment or fusion protein) thereof, including but not limited torabbits, mice, rats, sheep, goats, etc. In one embodiment, the PAMG-1polypeptide or fragment thereof can be conjugated to an immunogeniccarrier, e.g., bovine serum albumin (BSA) or keyhole limpet hemocyanin(KLH). Various adjuvants may be used to increase the immunologicalresponse, depending on the host species, including but not limited toFreund's (complete and incomplete), mineral gels such as aluminumhydroxide, surface active substances such as lysolecithin, pluronicpolyols, polyanions, peptides, oil emulsions, keyhole limpethemocyanins, dinitrophenol, and potentially useful human adjuvants suchas BCG (bacille Calmette-Guerin) and Corynebacterium parvum.

For preparation of monoclonal antibodies directed toward the PAMG-1polypeptide, or fragment, analog, or derivative thereof, any techniquethat provides for the production of antibody molecules by continuouscell lines in culture may be used. These include but are not limited tothe hybridoma technique originally developed by Kohler and Milstein(Nature 1975, 256:495-497), as well as the trioma technique, the humanB-cell hybridoma technique (Kozbor et al., Immunology Today 1983, 4:72;Cote et al., Proc. Natl. Acad. Sci. U.S.A. 1983, 80:2026-2030), and theEBV-hybridoma technique to produce human monoclonal antibodies (Cole etal., in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc.,pp. 77-96, 1985). In an additional embodiment of the present disclosure,monoclonal antibodies can be produced in germ-free animals(International Patent Publication No. WO 89/12690, published 28 Dec.1989). In fact, according to the present disclosure, techniquesdeveloped for the production of “chimeric antibodies” (Morrison et al.,J. Bacteriol. 1984, 159:870; Neuberger et al., Nature 1984, 312:604-608;Takeda et al., 1985, Nature 314:452-454) by splicing the genes from amouse antibody molecule specific for an PAMG-1 polypeptide together withgenes from a human antibody molecule of appropriate biological activitycan be used; such antibodies are within the scope of this presentdisclosure. Such human or humanized chimeric antibodies are preferredfor use in therapy of human diseases or disorders (described infra),since the human or humanized antibodies are much less likely thanxenogenic antibodies to induce an immune response, in particular anallergic response, themselves.

According to the present disclosure, techniques described for theproduction of single chain antibodies (U.S. Pat. Nos. 5,476,786 and5,132,405 to Huston; U.S. Pat. No. 4,946,778) can be adapted to producePAMG-1 polypeptide-specific single chain antibodies. Indeed, these genescan be delivered for expression in vivo. An additional embodiment of thedisclosure utilizes the techniques described for the construction of Fabexpression libraries (Huse et al., Science 1989, 246:1275-1281) to allowrapid and easy identification of monoclonal Fab fragments with thedesired specificity for a PAMG-1 polypeptide, or its derivatives, oranalogs.

Antibody fragments that contain the idiotype of the antibody moleculecan be generated by known techniques. For example, such fragmentsinclude but are not limited to: the F(ab)₂ fragment which can beproduced by pepsin digestion of the antibody molecule; the Fab fragmentswhich can be generated by reducing the disulfide bridges of the F(ab)₂fragment, and the Fab fragments which can be generated by treating theantibody molecule with papain and a reducing agent.

In the production of antibodies, screening for the desired antibody canbe accomplished by techniques known in the art, e.g., radioimmunoassay,ELISA (enzyme-linked immunosorbant assay), “sandwich” immunoassays,immunoradiometric assays, gel diffusion precipitin reactions,immunodiffusion assays, in situ immunoassays (using colloidal gold,enzyme or radioisotope labels, for example), Western blots,precipitation reactions, agglutination assays (e.g., gel agglutinationassays, hemagglutination assays), complement fixation assays,immunofluorescence assays, protein A assays, and immunoelectrophoresisassays, etc. In one embodiment, antibody binding is detected bydetecting a label on the primary antibody. In another embodiment, theprimary antibody is detected by detecting binding of a secondaryantibody or reagent to the primary antibody. In a further embodiment,the secondary antibody is labeled. Many means are known in the art fordetecting binding in an immunoassay and are within the scope of thepresent disclosure. For example, to select antibodies which recognize aspecific epitope of a PAMG-1 polypeptide, one may assay generatedhybridomas for a product which binds to a PAMG-1 polypeptide fragmentcontaining such epitope. For selection of an antibody specific to aPAMG-1 polypeptide from a particular species of animal, one can selecton the basis of positive binding with PAMG-1 polypeptide expressed by orisolated from cells of that species of animal.

In certain aspects disclosed herein, the PAMG-1-specific monoclonalantibodies disclosed herein can be, e.g., M271, produced by hybridomaN271, deposited with the Russian National Collection of IndustrialMicroorganisms (VKPM) Depository and assigned accession number VKPM-93;M52, produced by hybridoma N52, deposited with the VKPM and assignedaccession number VKPM-92; and M42, produced by hybridoma N42, depositedwith the VKPM and assigned accession number VKPM-94. The bindingproperties and other characteristics of those PAMG-1 specific monoclonalantibodies are disclosed in detail in U.S. Pat. No. 7,709,272 to Fuks etal. Hybridoma cell lines producing, e.g., PAMG-1 specific antibodies,such as those disclosed above, can be produced by the followingprocedure. First, mice having spleen and lymph node B-cells areimmunized with PAMG-1. Hybridomas are then produced to immortalize theB-cells. The B-cells may be spleen and/or lymph node B-cells. Thosehybridomas, which produce a monoclonal antibody having a bindingaffinity for PAMG-1, are then identified in an ELISA: first layer:PAMG-1; second layer: hybridoma supernatant; and third layer: conjugateof rabbit anti-mouse antibodies labeled by horse radish peroxidase.These identified hybridomas are then cultivated in vitro or in ascitesand the monoclonal antibodies they produce are isolated.

As disclosed herein, two or more PAMG-1 specific antibodies (e.g.,monoclonal antibodies) can be used in combination to detect PAMG-1 in avaginal fluid sample. In certain embodiments, at least one of theantibodies used in a method disclosed herein is detectably labeled. Avariety of detectable markers can be used, including, but not limitedto, stained particles, enzymes, fluorescent dyes, and radioactiveisotopes. One particular example of a detectable marker is a goldstained particle having an average dimension in the range of 20 to 30nm. Another example of a detectable marker is horseradish peroxidase.Methods for attaching a detectable marker to an antibody are described,for example, in Methods In Enzymology, 1981, Vol. 73, pp. 3-46 byHarlow, E., and Lane, D.; in “Antibodies a Laboratory Manual,” ColdSpring Harbor Laboratory, 1988, pp. 322, 323, and 343; and PierceCatalog, pp. T9-T17 (1996). Suitable enzymes include, but are notlimited to, alkaline phosphatase and horseradish peroxidase. Othermarkers or labels for use according to the present disclosure includecolloidal gold, colored latex beads, magnetic beads, fluorescent labels(e.g., fluorescene isothiocyanate (FITC), phycoerythrin (PE), Texas red(TR), rhodamine, free or chelated lanthanide series salts, especiallyEu3+, to name a few fluorophores), chemiluminescent molecules,radio-isotopes (¹²⁵I, ³²P, ³⁵S, chelated Tc, etc.) or magnetic resonanceimaging labels. Other markers include fluorescence quenching andfluorescence transfer markers, e.g., as used in homogenous as well assolid phase assays. Furthermore, in accordance with the presentdisclosure a marker can be an epitope, binding partner, or “handle” forinteraction with another molecule, such as biotin-streptavidin;glutathione-GST; hexahistidine-nickel; etc. The present disclosure alsocontemplates using secondary antibodies, which are themselves detectablylabeled, as markers (e.g., in a situation where the anti-PAMG-1 antibodypair uses antibodies with Fc portions from two different animalspecies).

The antibodies disclosed herein can be mobilizable (e.g., able to moveupon introduction of a fluid sample (e.g., in a flow device) and/orimmobilized (e.g., in the test region of a strip device). Methods forimmobilizing antibodies are well known in the art.

Detection of PAMG-1

Immunoassays, particularly immunochromatographic assays, constitute apreferred technique in accordance with the present disclosure, andimmunoassays are set forth in detail below. These assays have theadvantage of specificity, accuracy, speed, and economy. Other methodsfor detecting and quantitating PAMG-1, however, can also be used. Onesuch technique is mass spectrometry, e.g., using matrix-assistedlaser-desorption (MALDI) time-of-flight (TOF) mass spectrometry (MS)with delayed extraction and a reflectron in the time-of-flight chamber.Preferably MALDI assays are performed on silicon arrays. An example ofan array for MALDI is 200 μm circular gel pads at 350 μm centers, onoxidized silicon. A hydrophobic surface (repellent surface) betweengelpads further provides a more focused matrix/protein spot for MALDI,thereby improving signal for quantitation. For example, spots producedusing the Packard Bioscience system can be less than 200 μm in diameter.The Piezo system can deliver about 300 pL of MALDI matrix (e.g., DHB,sinapinic acid) to the exact position of the affinity captureagent-peptide spot to create a homogeneous peptide/matrix crystal.Desorption/Ionization (Karas, et al. Ion Processes, 1987, v. 78, pp.53-68 or Zenobi, et al. Mass Spectrom. Rev. 1998, v. 17, pp. 337-366)from this crystal in a MALDI-MS (e.g., Perseptive Voyager) yields a massspectrum where the height of a peptide peak is relative to the amountprotein containing that peptide.

An alternative technique for use in the methods disclosed herein iscapillary electrophoresis chromatography, which can permit quantitationof an analyte present in a small amount of sample.

Furthermore, quantitative biochemical techniques, such as polyacrylamidegel electrophoresis, high performance liquid chromatography, and thelike may be employed, alone or in combination, to detect and quantitatethe amount of PAMG-1 in a sample.

Such immunoassays using exemplary PAMG-1 specific antibodies encompassedby the presently disclosed methods are described in detail in U.S. Pat.No. 7,709,272 by Fuks et al.

Immunological Methods and Devices for Detecting PAMG-1

Various means known in the art for detecting immunospecific binding ofan antibody to an antigen can be used to detect the binding inaccordance with the present disclosure. An early method of detectinginteraction between an antigen and an antibody involved in analysis ofthe complex is by precipitation in gels. A further method of detectingan analyte-detector antibody binding pair includes the use ofradioiodinated detector antibodies or a radioiodinated protein which isreactive with IgG, such as Protein A. These early methods are well knownto persons skilled in the art, as reviewed in Methods in Enzymology,1980, v. 70, pp. 166-198. By selecting an antibody and conditions thatyield a positive result above the threshold values for PROM disclosedherein, one may employ this technology in the practice of the methodsdisclosed herein.

Later methods for determining the presence of an analyte in a sampleusing only one antibody included competitive binding assays. In thistechnique the antibody, which most often would be immobilized onto asolid support, would be exposed to a sample suspected of containing theanalyte together with a known quantity of labeled analyte. The twoanalytes, the labeled analyte and the analyte in the sample would thencompete for binding sites on the antibody. Either free labeled analyteor bound labeled analyte is determined, and from this measurement theamount of competing analyte in the sample is known. A more completedescription of this method is disclosed in “Basic Principles ofAntigen-Antibody Reaction”, Elvin A. Labat, (Methods in Enzymology, 70,3-70, 1980). In this example the labeled analyte can be labeled witheither a radioisotope or an enzyme label.

More current immunoassays utilize a double antibody method for detectingthe presence of an analyte. These techniques are also reviewed in theabove referenced volume of Methods in Enzymology. Therefore, accordingto one embodiment of the present disclosure, the presence of theindividual markers is determined using a pair of antibodies for each ofthe markers to be detected. One of said pairs of antibodies is referredto herein as a “detector antibody” and the other of said pair ofantibodies is referred to herein as a “capture antibody”. One embodimentof the present disclosure thus uses the double antibody sandwich methodfor detecting PAMG-1 in a sample of vaginal fluid. In this method, theanalyte is sandwiched between the detector antibody and the captureantibody, the capture antibody being irreversibly immobilized onto asolid support. The detector antibody would contain a detectable label,in order to identify the presence of the antibody-analyte sandwich andthus the presence of the analyte.

Common early forms of solid supports include plates, tubes or beads ofpolystyrene, all of which are well known in the field ofradioimmunoassay and enzyme immunoassay. More recently, a number ofporous materials such as nylon, nitrocellulose, cellulose acetate, glassfibers and other porous polymers have been employed as solid supports.

Thus, in a specific embodiment, the device of the disclosure comprisesmeans for conducting an immunochromatographic assay(“immunochromatographic assay device”). Such a device comprises a solidphase means for conducting a liquid. As used herein, the term “solidphase means for conducting a liquid” refers to a solid support thatallows migration of a liquid therethrough, e.g., via capillary action. Atypical product of this nature is a nitrocellulose membrane, which maybe prepared by methods well known to those skilled in the art.

Many immunochromatographic assay means and formats are known in the art,and can be used in the practice of the methods disclosed herein.Immunochromatographic assays using a membrane as a solid support in adipstick or flow-through device are well established for use in theclinical laboratory and for alternative, i.e., non-laboratory, sitetesting. The usual presentation for an immunochromatographic assaydevice is a membrane (cellulosic or non-cellulosic) enclosed in aplastic holder. The plastic holder keeps the membrane in a suitableconfiguration in order to ensure correct functioning of the entiredevice. There are many variations of the basic structure of assaydevices. For example, Litman et al. (U.S. Pat. Nos. 5,156,952 and5,030,558) describe an assay method and device for determining thepresence of a minimum amount of an analyte in a sample. Ullman et al.(U.S. Pat. Nos. 5,137,808 and 4,857,453) describe a device to house anassay membrane that includes self-contained liquid reagents to aidsample flow. Dafforn et al. (U.S. Pat. No. 4,981,768) describes a devicewith ports for applying sample and extra liquid. Corti et al. (EuropeanPatent Application No. 89118378.2), Greenquist et al. (U.S. Pat. No.4,806,312) and Berger et al. (U.S. Pat. No. 5,114,673) also describeassay devices.

Preferably, the immunochromatographic assay means includes a control toindicate that the assay has proceeded correctly. The control can be aspecific binding reactant at a spot more distal from the sampleapplication point on the solid phase support than the detection zonethat will bind to labeled reagent in the presence or absence of analyte,thus indicating that the mobilizable receptor has migrated a sufficientdistance with the liquid sample to give a meaningful result.

Suitable labels for use in immunochromatographic assays include enzymes,fluorophores, chromophores, radioisotopes, dyes, colloidal gold,colloidal carbon, latex particles, and chemiluminescent agents. When acontrol marker is employed, the same or different labels may be used forthe receptor and control marker.

One embodiment of the present disclosure uses a flow-through typeimmunoassay device. Valkirs et al. (U.S. Pat. No. 4,632,901) discloses adevice comprising antibody, specific to an antigen analyte, bound to aporous membrane or filter to which is added a liquid sample. As theliquid flows through the membrane, target analytes bind to the antibody.The addition of the sample is followed by the addition of a labeledantibody. The visual detection of the labeled antibody provides anindication of the presence of the target analyte in the sample.

Another example of a flow-through device is disclosed by Kromer et al.(EP-A 0 229 359), which describes a reagent delivery system comprising amatrix saturated with a reagent or components thereof dispersed in awater soluble polymer for controlling the dissolution rate of thereagent for delivery to a reaction matrix positioned below the matrix.

In migration type assays, the solid phase support, e.g., membrane, isimpregnated with the reagents needed to perform the assay. An analytedetection zone is provided in which labeled analyte is bound and theresults of the assay are read. For example, see Tom et al. (U.S. Pat.No. 4,366,241), and Zuk (EP-A 0 143 574). Migration assay devicesusually incorporate within them reagents that have been attached tocolored labels such as colloidal gold or carbon, thereby permittingvisible detection of the assay results without addition of furthersubstances. See for example, Bernstein (U.S. Pat. No. 4,770,853), May etal. (WO 88/08534), and Ching et al. (EP-A 0 299 428). All of these knowntypes of flow-through devices can be used according to the methodsdisclosed herein.

Direct labels are one example of labels that can be used inimmune-chromatographic assays according to the present disclosure. Adirect label has been defined as an entity, which in its natural state,is readily visible, either to the naked eye, or with the aid of anoptical filter and/or applied stimulation, e.g., U. V. light, to promotefluorescence. Examples of colored labels that can be used according tothe present disclosure, include metallic sol particles, for example,gold sol particles such as those described by Leuvering (U.S. Pat. No.4,313,734); dye sol particles such as described by Gribnau et al. (U.S.Pat. No. 4,373,932) and May et al. (WO 88/08534); dyed latex such asdescribed by May, supra, Snyder (EP-A 0 280 559 and 0 281 327); or dyesencapsulated in liposomes as described by Campbell et al. (U.S. Pat. No.4,703,017). Other direct labels include a radionuclide, a fluorescentmoiety or a luminescent moiety. In addition to these direct labelingdevices, indirect labels comprising enzymes can also be used accordingto the present disclosure. Various types of enzyme linked immunoassaysare well known in the art, for example, alkaline phosphatase andhorseradish peroxidase, lysozyme, glucose-6-phosphate dehydrogenase,lactate dehydrogenase, urease, these and others have been discussed indetail by Eva Engvall in Enzyme Immunoassay ELISA and EMIT in Methods inEnzymology, 70. 419-439, 1980 and in U.S. Pat. No. 4,857,453.

In a specific embodiment, the diagnostic device of the presentdisclosure comprises a membrane assembly having a detection sectionproximal to the point of introduction of the sample, and a capturesection downstream from that position. The detector section containsantibodies (detector antibodies) (e.g., monoclonal antibodies), whichwill react with any analytes of the present disclosure that are presentin the sample. The detector antibodies are reversibly immobilized ontothe membrane and will migrate with the sample, when in use. It ispreferred although not essential, that the detector antibodies arelabeled, for example, with a radionuclide, an enzyme, a fluorescentmoiety, luminescent moiety or a colored label such as those described inthe prior art, and discussed above. Specifically, one could employ areactive label, so that for example, the antibody would appear goldbefore capture of the antigen, and would change to purple upon capture.

The capture section which, as stated, is downstream from the detectorsection, comprises capture antibodies (e.g., monoclonal antibodies),which are irreversibly immobilized onto the solid support, each antibodyimmobilized at a different position in the capture section. Theantibodies and necessary reagents are immobilized onto the solid supportusing standard art recognized techniques, as discussed in theflow-through type immunoassay devices discussed previously. In general,the antibodies absorbed onto the solid supports as a result ofhydrophobic interactions between non-polar protein substructures andnon-polar support matrix material.

A particular advantage of the immunochromatographic assay technology ofthe present disclosure is that it overcomes the inability of theseassays to provide quantitative data. Thus, the capture section cancontain a mixture of immobilized antibodies specific for PAMG-1, suchthat a signal is only produced when the amount of PAMG-1 in the sampleexceeds the desired detection threshold.

In addition, the present disclosure contemplates use of homogeneousimmunoassay formats. One example of such a competitive homogeneousmethod is found in U.S. Pat. No. 3,817,837 by Rubenstein and Ullman,which describes a technique in which ligand and enzyme-bound-ligandcompete for antibody binding sites. Since binding of the antibody to theenzyme-bound-ligand alters its enzymatic activity, the concentration ofligand present can be estimated by measuring the rate at which such amixture converts substrate to product. Thus, in a homogeneous method,the detectable property of the label is inherently different dependingon whether bound or unbound. In its bound state, the label will havegreater or lesser signal intensity. Usually, binding of antibody to thelabeled ligand causes a decrease in signal intensity, e.g., when thelabel is an enzyme. Typical products in this category include the EMITline of enzyme immunoassays from Syva Company and the TDX line offluorescence polarization immunoassays from Abbott Diagnostics. Aparticular homogeneous assay could be prepared with the disposition ofall of the analytes on beads, in which event the sample would beintroduced and the beads thereafter spun down and detected.

Other examples of biological diagnostic devices that can be usedaccording to the present disclosure include the devices described by G.Grenner, P.B. Diagnostics Systems, Inc., in U.S. Pat. Nos. 4,906,439 and4,918,025. The Grenner '439 device comprises a diagnostic test elementand a sample application unit comprising a fluid delivery element thatis characterized as having a layer with a plurality of grooves for thedelivery of the sample to the test element. Grenner '025 relates to adevice that includes a sample introducing means such as a membraneadjacent to which is positioned a capillary containing a fixed reagentand a waste liquid reservoir. Release of the fixed reagent from thecapillary completes the reaction after the sample is deposited, andexcess liquid is retained by the waste reservoir, so that the device isself-contained.

While the measurement with a membrane is preferred, it is to beunderstood that other techniques and corresponding sensor devices maylikewise be used in similar fashion to the above. There are currentlyavailable several types of automated assay apparatus, which canundertake an assay on a number of samples contemporaneously. Theseautomated assay apparatuses include continuous/random access assayapparatus. Examples of such systems include OPUS™ of PB DiagnosticSystem, Inc. and the IMX™ Analyzer introduced by Abbott Laboratories ofNorth Chicago, Ill. in 1988. In general, a sample of the test fluid istypically provided in a sample cup and all the process steps includingpipetting of the sample into the assay test element, incubation andreading of the signal obtained are carried out automatically. Theautomated assay systems generally include a series of workstations eachof which performs one of the steps in the test procedure. The assayelement may be transported from one workstation to the next by variousmeans such as a carousel or movable rack to enable the test steps to beaccomplished sequentially. The assay elements may also includereservoirs for storing reagents, mixing fluids, diluting samples, etc.The assay elements also include an opening to permit administration of apredetermined amount of a sample fluid, and if necessary, any otherrequired reagent to a porous member. The sample element may also includea window to allow a signal obtained as a result of the process steps,typically a fluorescent or a colorimetric change in the reagents presenton the porous member to be read, such as by a means of a spectroscopy orfluorimeter, which are included within the assay system. The automatedassay instruments of PB Diagnostic Systems, Inc. are described in U.S.Pat. Nos. 5,051,237; 5,138,868; 5,141,871 and 5,147,609.

Further classes of immunochemical analyzer systems, which can be used inpracticing the methods disclosed herein, are the biosensors or opticalimmunosensor systems. In general an optical biosensor is a device thatuses optical principles quantitatively to convert chemical orbiochemical concentrations or activities of interest into electricalsignals. These systems can be grouped into four major categories:reflection techniques; surface plasmon resonance; fiber optic techniquesand integrated optic devices. Reflection techniques includeellipsometry, multiple integral reflection spectroscopy, and fluorescentcapillary fill devices. Fiber-optic techniques include evanescent fieldfluorescence, optical fiber capillary tube, and fiber optic fluorescencesensors. Integrated optic devices include planer evanescent fieldfluorescence, input grading coupler immunosensor, Mach-Zehnderinterferometer, Hartman interferometer and difference interferometersensors. Holographic detection of binding reactions is accomplisheddetecting the presence of a holographic image that is generated at apredetermined image location when one reactant of a binding pair bindsto an immobilized second reactant of the binding pair (see U.S. Pat. No.5,352,582, issued Oct. 4, 1994 to Lichtenwalter et al.). Examples ofoptical immunosensors are described in general in a review article by G.A. Robins (Advances in Biosensors), Vol. 1, pp. 229-256, 1991. Morespecific descriptions of these devices are found for example in U.S.Pat. Nos. 4,810,658; 4,978,503; and 5,186,897; R. A. Brady et al. (Phil.Trans. R. Soc. Land. B 316, 143-160, 1987) and G. A. Robinson et al. (inSensors and Actuators, Elsevier, 1992).

The methods and corresponding kits of the present disclosure are capableof incorporation and practice within a variety of optical measurementsystems. Specifically, while the kits and materials of the presentdisclosure may be practiced in an immunoassay format, such format itselfis capable of embodiment in a variety of optoelectronic detectionsystems. More particularly, a variety of optical immunosensortechnologies are already known that may be facilitated and implementedin the practice of the methods disclosed herein. Thus, for example,devices and techniques such as reflection techniques, surface plasmonresonance, fiber optic waveguide techniques and integrated opticdevices, may all be adopted and specifically configured to detect anddisplay the results of the examination of a patient's biological samplein accordance with the present method. Particular reflection techniques,such as reflectometry and ellipsometry, and the specific use of opticalfibers, optical waveguides, fluorescent capillary fill devices andintegrated optical biosensors, present but a few of the varianttechniques and equipment that may be employed. A general review of thesedevices may be found in Robinson, G. A., Optical Immunosensors: AnOverview, Advances in Biosensors, Vol. 1, pp. 229-256 (1991).

More particularly, ellipsometry relies on the direction of a polarizedlight beam first against a reference surface (a standard) and thereafteragainst the sample surface, following which a comparison of the natureand extent of the resulting reflections can be made. Particularly, thebinding of analyte to receptor molecules will be measured as a chain thethickness of the surface relative to the reference surface.

In the instance of multiple internal reflection spectroscopy, forexample, the ligand and its receptor may be covalently immobilized onthe optical surface of a planar, fused-quartz waveguide after which alight beam may be internally reflected within the waveguide and wouldpenetrate into a solution adjacent the waveguide, so that refractivedifferences would be capable of measurement as between the standard andthe sample. In this particular format, a fluorescent label may beassociated and measurements of fluorescence resultantly taken todetermine the present extent of binding.

An additional technique utilizes the technology known as fluorescentcapillary fill device. In this particular technology, two glass platesheld apart by a gap of capillary dimension are utilized. Receptormolecules may be immobilized onto the base plate, which also acts as anoptical waveguide. Competitive or sandwich assays utilizing FITClabeling may be performed and induced fluorescence is coupled into thewaveguide with signal from bound as opposed to unbound sources. Suchsignal is discriminated by its angular divergence upon exiting thewaveguide. Surface Plasmon Resonance (SPR) devices have also beenprepared which operate in response to the coupling of light incidentupon a thin metal film into surface modes associated with collectiveelectron oscillations within the metal film. Resonance condition isdependent upon the optical characteristics of the metal film, itsthickness, the refractive indices of the dielectric on either side ofit, and the angle of incidence of light. Receptor molecules are bound tothe top side of the metal film, and the light is directed at the bottomside of the film, such as through a prism substrate. The target analyte,when binding to these receptors, will cause a shift in the resonancecondition because of the change it produces in the local refractiveindex. Resonance is observed by a monitoring of the reflected lightintensity as the angle of incidence at the light beam on the metal filmsurface varies. The change in resonance angle will directly correlatewith the amount of analyte bound.

The techniques involving fiber optic systems include the evanescentfield fluorescence. In this instance, the cladding is removed from theend of an optical fiber, thus producing a sensor element thatevanescently interacts with the surrounding medium. Receptor moleculesare bound to the exposed fiber surface, and direct assays may beperformed utilizing the natural fluorescence of the receptor andconjugate proteins. Competitive or sandwich assays may be performedusing FITC labeling to achieve greater sensitivity. In operation, alight wave is coupled into the fiber, and a portion of the evanescentlyproduced fluorescence is coupled back into the fiber and propagated backto a detector.

A further technique utilizing optical fiber technology involves theoptical fiber capillary tube, in which a bare fiber optic is enclosedwithin a cylindrical fill chamber, producing a sensor element thatinteracts evanescently with the portion of the fill volume immediatelysurrounding the fiber. Receptor molecules may be bound to the exposedfiber surface and sandwich or competitive displacement assays may beperformed. A light wave would be coupled into the fiber, and a portionof the evanescently induced fluorescence would be coupled back into thefiber and propagated back to a detector. The signal from the targetanalyte versus the background sources is discriminated by its angulardivergence upon exiting the fiber. Other fiber optic techniques such asfiber optic fluorescence may be adapted to the methods disclosed hereinutilizing certain of the same principles enunciated above.

Further photonic techniques such as interferometry include thedisposition of a thin-film waveguide having, for example, two paths, onthe first of which receptor molecules may be immobilized while thesecond is shielded to provide a reference channel. Laser light, forexample, may be coupled into the waveguide and split down the two paths,so that changes in the refractive index and thickness of the coveringletter may be detected by the result of a phase shift in the beam, whichwill, in turn, correlate with the amount of analyte bound. A variationon this approach is identified in the Hartman interferometer, where asingle path multimode thin film planar waveguide is prepared. Receptormolecules may be immobilized on this path, and light from a laser may becoupled into the waveguide so that two modes propagate down the path.The optics of multimode geometries are such that the higher order modehas a large evanescent field, providing a signal mechanism, and thelower order mode has practically no evanescent field, providing areference mechanism. Binding with the target analyte will cause relatedchanges in the refractive index and thickness of the covering layer overthe path which will be detected by the evanescent field of the higherorder mode, causing a phase shift in that mode. As the lower order orreference mode is blind to such changes, no phase shift will beexperienced, and the measured difference between the signal andreference beams will be capable of correlation to determine the amountof analyte bound.

While the foregoing discussion has provided both in general terms andsome detail, various techniques available in optical sensor technologyare adaptable to the practice of the present disclosure. It is to beunderstood that the above recitation is by no means exhaustive orlimitative, as a variety of extant technologies may be adopted, thatwill successfully measure differences in binding and, consequently, thepresence and amount of the respective markers or analytes of interestherein. Of course, as emphasized above, no matter what technology isemployed, the practice of the methods disclosed herein comprisessimultaneous detection and measurement of at least three analytes.

Immunochromatographic Methods for Detecting PAMG-1

Embodiments of the methods of detecting PAMG-1 according to the presentdisclosure are described below.

In one embodiment of the method, PAMG-1 is detected in a sample throughthe contact of a sample containing PAMG-1 with an immunoassay systemaccording to the methods disclosed herein to form an antibody-PAMG-1complex. The antibody-PAMG-1 complex is then detected. In one variationof this embodiment, the antibody includes a detectable marker, the stepof detecting the antibody-PAMG-1 complex, which includes the detectablemarker.

In another embodiment of the method, PAMG-1 is detected in a sample byputting the sample in contact with an antibody which has a highlyspecific binding affinity for PAMG-1 (like M271, exemplified infra),thus forming the antibody M271-PAMG-1 complex. The complex then comesinto contact with an immobilized second antibody (e.g., like M52). Thesecond antibody is immunologically distinct from the first antibody(e.g., binds to a different epitope), so that such antibodies cansimultaneously bind to the PAMG-1 molecule. The immobilized antibodybinds to the mobile antibody PAMG-1 complex to form the immobilizedantibody PAMG-1 antibody complex. PAMG-1 is detected by detecting thisheterotrimer complex. As noted above, the antibody with high specificityfor PAMG-1 is preferably used for the initial recognition of PAMG-1.

When the above-described method includes the use of one antibody of theselected pair labeled with a detectable marker, a variation of themethod includes putting the sample in contact with the first, labeledantibody prior to contact of the sample with the second, immobilizedantibody. In this variation, the labeled antibody serves to bind toPAMG-1 in the sample. Yet another embodiment of the method includes thefollowing steps: adding a fluid sample containing PAMG-1 to amobilizable, labeled antibody region of porous material which permitsmigration of antibodies and proteins therethrough, the antibody regionincluding a mobilizable antibody which has a high specificity for PAMG-1resulting in the attachment of the antibody to PAMG-1 to form anantibody PAMG-1 complex; migration of the complex to the test regioncontaining a second antibody immobilized therein, which second antibodyhas a binding affinity for PAMG-1 resulting in the second antibodybinding to the labeled antibody-PAMG-1 complex to form an immobilizedcomplex; and detecting the immobilized complex in the test region.

Yet another embodiment of the method is a standard sandwich assay, inwhich an unlabeled antibody is immobilized on any surface. Addition offluid sample containing PAMG 1 results in binding of PAMG-1 by theimmobilized antibody to form an antibody PAMG-1 complex. Addition oflabeled antibody results in formation of an immobilized complex composedof immobilized antibody PAMG-1-labeled antibody and detection of thiscomplex.

According to the above-described methods, the antibodies may include adetectable marker or label, the step of detecting the antibody-PAMG-1 orPAMG-1-antibody complex including detection of the detectable marker orlabel. Examples of detectable markers that can be used include stainedparticles, enzymes, dyes and radioactive isotopes. In a specificembodiment, the detectable marker is a stained particle of gold, e.g.,having an average dimension between about 20 nm and 30 nm. In yetanother embodiment, the detectable marker is horseradish peroxidase.

Exemplary Devices for Detecting PAMG-1

A variety of devices are envisioned for detecting PAMG-1 protein in asample. Devices and/or methods according to the present disclosurepreferably can detect PAMG-1 in a sample where the concentration ofPAMG-1 is between about 1 ng/ml and 50 μg/ml, about 2 ng/ml and 50μg/ml, about 3 ng/ml and 50 μg/ml, or about 4 ng/ml and 50 μg/ml.Non-limiting examples of devices that can be used in the methodsdisclosed herein are described in U.S. Pat. No. 7,709,272 by Fuks et al.Devices for use in the present methods also include, e.g., a cassettecontaining a test strip (e.g., with a pad region where the sample isplaced, and test region (where results are read)), and optionally, abuilt-in timer and/or a site to indicate patient identification. The padand test regions are discussed in more detail below. In certainembodiments of the present methods, the preferred detection threshold ofPAMG-1 is adjusted to be at least about 4 ng/ml. It is to be understoodthat the methods and devices of the present disclosure also encompassPAMG-1 detection thresholds of about at least 1 ng/ml, about at least 2ng/ml, and about at least 3 ng/ml.

The devices and methods described herein can be adapted to be usedeasily in a rapid and convenient manner, thereby making it possible forthe devices and methods to be used in outpatient conditions. Forexample, the method can be incorporated into an easy-to-use device thatcan be operated by a patient with little or no prior experience with thedevice. This makes the method and device highly reliable and not verysusceptible to operator error. The method can also be designed to enablea simple “yes” or “no” (or “+” or “−”) determination of the presence ofPAMG-1 in a sample (e.g. vaginal fluid sample).

An exemplary, non-limiting device for detecting PAMG-1 is illustrated inFIGS. 1 and 2. For purposes of exemplification, this description refersto monoclonal antibodies exemplified infra. However, it is not necessarythat these specific monoclonal antibodies be used. The procedure ofselection of, e.g., a pair of PAMG-1 specific antibodies, such as, e.g.,those described above, can be reproduced by an artisan of ordinary skillin the art.

As shown in FIGS. 1 and 2, an exemplary device that can be used toperform the methods disclosed herein has a strip-like body composed ofseveral sequentially interconnected elements. More specifically, part 12of the device comprises a pad, which contains M271 antibody region 10,in which the M271 antibodies are labeled, e.g., by stained particles SP(not shown in the drawings). Pad 12 may be made of a fiberglass tissueor any other material, which is porous and permits the migration ofvarious particles and substances of a sample. Stained particles maycomprise gold particles having an average dimension within the range of20 to 30 nm. The M271 antibody region also contains mouse IgGimmunoglobulin labeled by the same stained particles. The labeled M271antibodies and mouse IgG immunoglobulin are introduced into the bandpart 10 of pad 12 by impregnating pad 12 with a solution of labeled M271antibodies and labeled mouse IgG. The solution of M271 antibodies andmouse IgG immunoglobulin may be introduced in nitrocellulose membrane 22using drawing pen or microdrop forming device. Connected to one end ofpad 12 in its longitudinal direction are [a] nitrocellulose membrane 22,which contains a test region 14 and a control region 16. Both the testregion 14 and control region 16 are arranged transversely to the deviceover its entire width. Test region 14 is a band portion ofnitrocellulose membrane 22. Test region 14 contains M52 antibodiesattached to nitrocellulose membrane 22. Control region 16 containsanti-mouse anti immunoglobulin antibodies attached to nitrocellulosemembrane 22. Control region 16 crosses the entire width of strip 22. Afilter paper membrane 24 is connected to the end of nitrocellulosemembrane 22, which is opposite to the end of nitrocellulose membrane 22connected to pad 12. A filter paper membrane 24 is connected to the endof nitrocellulose strip 22 in its longitudinal direction. The surface ofthe device is coated with special protective films 28 and 30, e.g., thinadhesive tapes specially designed for strip devices. Arrows 18 are drawnon the surface of film 28 in order to show the sample application end ofpad 12. Pad 12, nitrocellulose membrane 22 and filter paper strip 24 areattached to an adhesive rigid plastic base 26.

In the embodiment described in this section, the device includes an M271antibody pad region 10 formed of a porous sample application matrix thatpermits migration of antibodies and proteins therethrough. The M271antibody region 10 includes the M271 antibody, which is capable ofhighly specific binding to PAMG-1. Introduction of fluid samplecontaining PAMG-1 into M271 antibody region results in the attachment ofthe M271 antibody to PAMG 1 to form the antibody M271-PAMG-1 complex.The device also includes a test region 14 in fluid connection with M271antibody region 10 formed of a porous material which permits migrationof antibodies and proteins therethrough. Test region 14 includes the M52antibody immobilized in test region 14 which is also capable of bindingto PAMG-1. The M52 antibody is immunologically distinct from the M271antibody such that the M271 and M52 antibodies can simultaneously bindto PAMG-1. Introduction of a fluid sample to the M271 antibody region 10results in the migration of the antibody M271-PAMG-1 complex into thetest region 14 where the antibody M271-PAMG-1 complex binds to the M52antibody and is immobilized in the test region by the M52 antibody. Thedevice detects PAMG-1 in a sample based on the presence of the M52antibody immobilized in test region 14. As a result, only PAMG-1 formsan antibody M271-PAMG-1-M52 antibody complex which is immobilized in thetest region 14. Thus, the presence of the M52 antibody immobilized inthe test region 14 is indicative of the presence of PAMG-1 in thesample.

In this embodiment of a device for detecting PAMG-1 in vaginalsecretions, the M271 antibody is attached to a detectable marker whichis used to detect PAMG-1 immobilized in the test region 14. Examples ofdetectable markers that may be used include, but are not limited to,stained particles, enzymes, dyes, fluorescent dyes, and radioactiveisotopes. In one embodiment, the detectable marker is gold particleshaving an average dimension between about 20-30 nm. In one embodiment,the M271 antibody is a labeled antibody in a freeze-dried state.

In a variation of the embodiment where the M271 antibody in the M271antibody pad region is labeled with a detectable marker, the devicefurther includes test region, which contains the M52 antibody. The padregion and test region are in fluid connection.

In yet another embodiment of the device, also embodied within the deviceillustrated in FIGS. 1 and 2, the device has a strip-like body withproximal and distal ends. The M271 antibody region 10 of the strip-likebody is made of a material which permits the migration of antibodies andproteins therethrough. The M271 antibody region 10 of the strip-likebody includes the M271 antibody, which has a highly specific bindingaffinity for PAMG-1, introduction to the M271 antibody pad region of afluid sample containing PAMG-1, which results in the attachment of theM271 antibody to PAMG-1 to form the antibody M271-PAMG-1 complex.

The strip-like body also includes a test region 14, which is proximal tothe M271 antibody region 10 and is in fluid connection with the M271antibody region 10. The test region 14 is formed of a material whichpermits migration of antibodies and proteins therethrough. The testregion 14 includes the M52 antibody immobilized in the test region 14,which has a binding affinity for PAMG-1, the introduction of the fluidsample to the M271 antibody region 10 resulting in the migration of theantibody M271-PAMG-1 complex to the test region 14 where the antibodyM271-PAMG-1 complex binds to the M52 antibody and is immobilized in testregion 14 by the M52 antibody. The test region can also include M42antibody and M52 antibody immobilized in the test region 14. The devicedetects PAMG-1 in a sample based on the immobilization of the complex oflabeled antibody M271-PAMG-1 in the test region 14. Using variouscombinations of PAMG-1 specific antibodies (e.g., M42 and M52)immobilized in the test region exemplifies one way to adjust thesensitivity threshold (detection threshold) of the strip device (seeU.S. Pat. No. 7,709,272 by Fuks et al.). However, the artisan ofordinary skill in the art will appreciate that other methods ofadjusting the detection threshold are possible (e.g., varying thebinding affinity of the immobilized and immobilizable antibodies of apair of PAMG-1 specific antibodies and/or adjusting the procedure, e.g.,the procedural timing of the steps of the testing procedure, asdisclosed herein).

Control Region. The device can include a standard control region 16(FIGS. 1 and 2). This control region serves to confirm the properoperation of the device. However, any alternative control-region designsmay also be used with a device for use in the methods disclosed herein.

For example, a device with one control region can include the M271antibody region 10 formed of a material which permits migration ofantibodies and proteins therethrough, the M271 antibody region 10including a labeled M271 antibody that is not immobilized therein andhas a high specificity for PAMG-1, introduction to the M271 antibody padregion 10 of a fluid sample containing PAMG-1 resulting in the M271antibody binding to PAMG-1 to form an antibody M271-PAMG-1 complex. Thedevice can also include a test region 14 in fluid connection with M271antibody region 10 which is formed of a material which permits migrationof antibodies and proteins therethrough. The test region 14 alsoincludes the M52 antibody immobilized in the test region 14 which has abinding affinity for PAMG-1. The M52 antibody is immunologicallydistinct from the M271 antibody such that the M271 and M52 antibodiescan simultaneously bind to PAMG-1. Introduction of the fluid sample tothe M271 antibody region 10 results in the migration of the antibodyM271-PAMG-1 complex into the test region 14 where the antibodyM271-PAMG-1 complex binds to the M52 antibody and is immobilized in testregion 14 by the M52 antibody. The device detects PAMG-1 in a samplebased on the immobilization of the labeled M271 antibody in the testregion 14. When a low concentration of PAMG-1 is present in the sample,at least some of the labeled M271 antibodies migrate from the M271antibody region 10 through the test region 14 to the control region 16.Anti-mouse anti-immunoglobulin antibodies are immobilized in the controlregion 16. Anti-immunoglobulin antibodies bind labeled M271 antibodiesthat stain the control region. If a high concentration of PAMG-1 ispresent in the sample, then only a low quantity of labeled M271antibodies can approach the control region 16 and coloration of thecontrol region may be too weak to become visible to the naked human eye.To prevent such a possibility, labeled mouse IgG immunoglobulin wasadded into M271 antibody region 10. This immunoglobulin does not bindPAMG-1 and migrates freely through M52 antibody test region 14 to thecontrol region 16 where it is bound by anti-mouse antiglobulinantibodies and stains control region 16. The control region confirms theproper functioning of the device regardless of the concentration ofPAMG-1 in the sample.

Yet another component of the device can be a porous material that is intight porous connection with material of test region. This part ofdevice works as a pump that helps to move liquids, proteins andantibodies therethrough. Examples of detectable markers, which may beused for the labeling of mouse antibodies and IgG immunoglobulininclude, but are not limited to stained particles, enzymes, dyes, andradioactive isotopes. In one embodiment, the detectable marker is afluorescent dye. In yet another embodiment, the detectable markers arestained particles. In one embodiment, the M271 antibody, which is alabeled antibody and the labeled mouse immunoglobulin IgG are in afreeze-dried state.

The materials used in the various regions of the above-described devicemay be any combination of materials that permit the migration ofantibodies and proteins therethrough. Examples of suitable materialsinclude but are not limited to fiberglass, porous plastic,nitrocellulose, and filter paper.

The parts of a device for use in a method disclosed herein can bepositioned in any functional combination (e.g., in a lateral flowdevice, cassette, etc.) provided that PAMG-1 can be detected in thesample when present at a concentration of at least a predefineddetection threshold (e.g., 4 ng/ml).

Devices for use in the present methods may optionally include aprotective film covering at least a portion of the device. It can betransparent or not transparent and can have necessary trademark,informational marks/signs or arrows on its surface.

Sample Collection

In the methods disclosed herein, it is necessary to collect a vaginalfluid sample from a patient. The device or tool or other means used tocollect the sample, and transfer the sample to solution (for testing),can be varied according to the present disclosure, so long as the sampleis collected. Non-limiting examples of devices for collecting vaginalfluid sample (e.g., a vaginal fluid sample containing PAMG-1), include,e.g., vaginal swabs (e.g., flocked vaginal swabs).

Non-limiting examples of other means to collect a vaginal fluid sampleinclude, e.g., douche method or vaginal wash. Also, a syringe may beused to collect the vaginal fluid sample.

The specific device and/or method of sample collection can be varied,and any suitable device or method known in the art can be used, so longas the vaginal fluid sample is successfully collected. Preferably, thedevice or means of sample collection yields at least 70%, at least 75%,at least 80%, at least 85%, at least 90%, at least 95%, or greater ofthe target analyze in the sample (e.g. PAMG-1). For example, the flockedvaginal swab used in the present Examples yields about 80-90% of thePAMG-1 after collection and transfer to solution.

In certain embodiments, the device or means used to collect the vaginalfluid sample provides a 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, or1:10 dilution of the vaginal fluid sample. In certain embodiments, thedevice of or means used to collect the vaginal fluid sample provides adilution of the vaginal fluid sample in the range of 1:1 to 1:10, 1:2 to1:9, 1:2 to 1:8, 1:2 to 1:7, 1:2 to 1:6, 1:2 to 1:5, 1:2 to 1:4, 1:2 to1:3, 1:3 to 1:10, 1:3 to 1:9, 1:3 to 1:8, 1:3 to 1:7, 1:3 to 1:6, 1:4 to1:7, or 1:5 to 1:6.

In a specific embodiment, a vaginal swab provides about a 1:4 dilutionof the vaginal fluid sample. In another embodiment, a flocked vaginalswab provides about a 1:4 dilution of the vaginal fluid sample.

Kits

The present disclosure also provides kits. In one aspect a kit disclosedherein comprises a device, e.g., as disclosed herein (e.g., a lateralflow device) for detecting the presence of PAMG-1 in a vaginal fluidsample when present at a level above a predetermined detection threshold(e.g., 0.5 ng/ml, 1 ng/ml, 2 ng/ml, 3 ng/ml, or 4 ng/ml). In anotheraspect a kit disclosed herein comprises a device (such as, but notlimited to a lateral flow device, e.g., such as a device similar to onedescribed in U.S. Pat. No. 7,709,272) for detecting the presence ofPAMG-1 in a vaginal fluid sample, when present at a level above apredetermined threshold (e.g., 0.5 ng/ml, 1 ng/ml, 2 ng/ml, 3 ng/ml, or4 ng/ml); and a means for collecting a vaginal fluid sample (e.g., avaginal swab, such as, but not limited to, a vaginal swab describedherein (e.g., a flocked vaginal swab), a syringe, a douche kit, or othersuitable device for collecting the sample). In certain aspects, themeans for collecting the vaginal fluid sample can optionally be used todilute the vaginal fluid sample (e.g., 1:1, 1:2, 1:3, 1:4, 1:5, 1:6,1:7, 1:8, 1:9, 1:10, or, e.g., in a range of 1:1 to 1:10, 1:2 to 1:9,1:2 to 1:8, 1:2 to 1:7, 1:2 to 1:6, 1:2 to 1:5, 1:2 to 1:4, 1:2 to 1:3,1:3 to 1:10, 1:3 to 1:9, 1:3 to 1:8, 1:3 to 1:7, 1:3 to 1:6, 1:4 to 1:7,or 1:5 to 1:6, etc.). The kits can also comprise a solvent fortransferring the vaginal fluid sample (e.g., containing an analyte,e.g., PAMG-1), e.g., a solution containing: 0.9% NaCl, 0.01% TritonX100, 0.05% NaN₃). The solvent can be contained within a vial that canalso be used as applicator of the solvent plus vaginal fluid sample canbe applied directly onto a lateral flow device. A kit can also comprisea cassette containing a test strip (e.g., with a pad region where thesample is placed, and test region (where results are read)), andoptionally, a built-in timer and/or a site to indicate patientidentification. The kits disclosed herein can further comprise one ormore vials (e.g., plastic vial) and/or instructions for use. Forexample, instructions for use can include directions for diagnosing TTDbased on the results of the test. The kits can also comprise adesiccant. A kit can also comprise a timer, e.g., built in to the testdevice, or as a separate unit. The instructions for use can in addition,or alternatively, contain instructions for diagnosing risk ofspontaneous rupture of fetal membranes (ROM) (e.g., preterm prematureROM) and/or risk of preterm delivery. The kits can comprises a device asillustrated in FIGS. 1 and 2.

Sample Collection and Test Procedure

In general, the methods and kits disclosed herein can be used to collectspecimens (vaginal fluid samples) from patients presenting with signs,symptoms or complaints suggestive of preterm labor. Preferably, thespecimen is collected prior to digital examination or lubricants, andprior to use of any disinfectant solutions or medicines or 6 hours aftertheir removal. The specimen can be collected in the presence ofnon-significant blood admixtures. The methods disclosed herein can beperformed even with trace amounts of blood on the collection device(e.g., swab). The specimen can also be collected if urine, semen orvaginal infections are present, and can be collected from patients from20 to 36 weeks, 6 days, gestational age. Further, speculum examinationis not required.

The following methods for collecting a vaginal fluid sample from apregnant woman and assaying the sample for the presence of PAMG-1 (e.g.,for the prediction of TTD and/or for determining a patient's risk ofpreterm labor and/or spontaneous ROM) according to the presentdisclosure can be used. Although the skilled artisan will appreciatethat different methods and/or test devices can be used to achieve thesame results, as disclosed herein, and are also encompassed by thepresent disclosure.

Sample Collection

In one example of a test according to the present methods, a sample ofcervico-vaginal discharge collected by vaginal swab is extracted into asolvent as follows:

Take the solvent (e.g., containing: 0.9% NaCl, 0.01% Triton X100, 0.05%NaN₃) vial by its cap and shake well to make sure all liquid in the vialhas dropped on the bottom. Open the solvent vial and put it in avertical position. To collect a sample from the surface of the vagina, avaginal swab can be used (e.g., a sterile flocked swab), or othersuitable collection device or means to collect the vaginal fluid sample,as disclosed above. For vaginal swab, the swab tip should not touchanything prior to its insertion into vagina. Hold the swab in the middleof the stick and, while the patient is lying on her back, carefullyinsert the swab tip of the swab into the vagina until the fingerscontact the skin no more than about 2-3 inches (5-7 cm) deep. Withdrawthe swab from the vagina after about 30 seconds (other lengths of time,e.g., about 10, 20, 40, 50, 60, 90, 120 seconds, 3 minutes, 4 minutes, 5minutes, etc.). Place the swab tip into the vial and rinse the swab inthe solvent (e.g., 0.55 ml solvent) by rotating for about 30 seconds(other lengths of time, e.g., about 10, 20, 40, 50, 60, 90, 120 seconds,3 minutes, 4 minutes, 5 minutes, etc.). Remove and dispose of the swab.The skilled artisan will appreciate that the above procedure and samplecollection and transfer of sample to solvent times may vary if otherdevice(s) and/or means or methods are used to collect the vaginal fluidsample. Other such devices and procedures and procedural timing are alsoencompassed by the present methods.

Test Procedure (PAMG-1 Detection)

Following transferring the vaginal fluid sample obtained from thepatient to solution (e.g., by rinsing of the swab in the solvent),contact a PAMG-1 test device, e.g., as disclosed herein, e.g., a lateralflow device, with the solvent. In one embodiment, the sample flows froman absorbent pad to a nitrocellulose membrane, passing through areactive area containing monoclonal anti-PAMG-1 antibodies conjugated toa gold particle. The antigen-antibody complex flows to the test regionwhere it is immobilized by a second anti-PAMG-1 antibody. This eventleads to the appearance of a test line. Unbound antigen-antibodycomplexes continue to flow along the test strip and are immobilized by asecond antibody. This leads to the appearance of an internal controlline. In one embodiment, the test strip is dipped into the vial withsolvent for about 5 minutes (other lengths of time, e.g., about 1, 2, 3,4, 6, 7, 8, 9, 10 minutes, are also contemplated herein, depending uponthe specific conditions of the test and the specific method or deviceused to test the sample). The test strip can be removed as soon as twostripes are clearly visible in the vial (about 5 minutes). The resultscan then be read (e.g., by placing the test on a clean, dry, flatsurface). In one embodiment, the presence of two lines indicates apositive test result (PAMG-1 detected) and the presence of one lineindicates a negative result. The skilled artisan will appreciate thatthe above procedural steps and timing are exemplary only, and are notlimiting.

As discussed above, it is to be understood that variations of thisprocedure are also encompassed by the present disclosure, so long asthey result in the detection of PAMG-1 in the vaginal fluid sample whenpresent at a predefined detection threshold (e.g., about at least 4ng/ml, about at least 3 ng/ml, about at least 2 ng/ml, or about at least1 ng/ml). Thus, for example, the type and volume of solvent, device ormeans for sample collection, and PAMG-1 detection device can be variedor completely different from those disclosed as examples herein. Theincubation times above, e.g., 30 second sample collection with swab, 30second rinse of swab in solvent may vary depending on the specificprocedure and test device used. The site of vaginal fluid samplecollection can vary, and can be determined by one of ordinary skill inthe art. By way of non-limiting example, exemplary sites of collectionof vaginal fluid samples include collection from, e.g., cervical os,cervical canal, posterior fornix, vaginal cavity/canal. Collection ofthe sample can be blind (i.e., collected from the vagina without use ofa speculum).

In accordance with the present disclosure, there may be employedconventional molecular biology, microbiology, recombinant DNA,immunology, cell biology and other related techniques within the skillof the art. See, e.g., Sambrook et al., (2001) Molecular Cloning: ALaboratory Manual. 3rd ed. Cold Spring Harbor Laboratory Press: ColdSpring Harbor, N.Y.; Sambrook et al., (1989) Molecular Cloning: ALaboratory Manual. 2nd ed. Cold Spring Harbor Laboratory Press: ColdSpring Harbor, N.Y.; Ausubel et al., eds. (2005) Current Protocols inMolecular Biology. John Wiley and Sons, Inc.: Hoboken, N.J.; Bonifacinoet al., eds. (2005) Current Protocols in Cell Biology. John Wiley andSons, Inc.: Hoboken, N.J.; Coligan et al., eds. (2005) Current Protocolsin Immunology, John Wiley and Sons, Inc.: Hoboken, N.J.; Coico et al.,eds. (2005) Current Protocols in Microbiology, John Wiley and Sons,Inc.: Hoboken, N.J.; Coligan et al., eds. (2005) Current Protocols inProtein Science, John Wiley and Sons, Inc.: Hoboken, N.J.; Enna et al.,eds. (2005) Current Protocols in Pharmacology John Wiley and Sons, Inc.:Hoboken, N.J.; Hames et al., eds. (1999) Protein Expression: A PracticalApproach. Oxford University Press: Oxford; Freshney (2000) Culture ofAnimal Cells: A Manual of Basic Technique. 4th ed. Wiley-Liss; amongothers. The Current Protocols listed above are updated several timesevery year.

The following examples are meant to illustrate, not limit, the presentdisclosure.

EXAMPLES Example 1: PAMG-1 Detection Kit (TTD Test)

A kit for the detection of PAMG-1 at a detection threshold of 4 ng/mlwas prepared. The kit included a diagnostic device employing monoclonalantibodies that detect PAMG-1 present in cervico-vaginal secretions, asdescribed in detail in U.S. Pat. No. 7,709,272 by Fuks et al. Thediagnostic device is illustrated in FIGS. 1 and 2. The diagnostic deviceitself can detect PAMG-1 when present at a concentration of at least 1ng/ml in the sample. The kit also included a flocked vaginal swab withthe following specifications: length of the plastic shaft: 170.0 mm±1mm; plastic tip diameter: 4.6 mm±0.1 mm; stick diameter handle part: 4.4mm±0.2 mm; length of the fibre tip: 22 mm±3 mm; flocked tip diameter:7.00 mm±1.5 mm; total length: 171 mm±2 mm. The kit further includedinstructions for sample collection and the testing procedure. The samplecollection and testing procedure included a 30 second swab saturation inthe vagina (a sterile speculum examination was not required), a 30second active washing step whereby the swab just removed from the vaginawas actively rotated in a solvent filled vial and a 5 minute waitingperiod from the time the swab was removed and the test strip wasinserted if two testing lines did not appear sooner. During the testprocedure, PAMG-1 present in the sample sequentially bound to monoclonalantibody conjugated with labeled particles, then to monoclonal antibodyimmobilized on an insoluble carrier. The in vivo sensitivity detectionthreshold of PAMG-1 was adjusted to 4 ng/ml using the specific samplecollection and TTD test procedure described below:

Sample Collection and TTD Test Procedure:

-   -   1. Take the solvent (containing: 0.9% NaCl, 0.01% Triton X100,        0.05% NaN₃) vial by its cap and shake well to make sure all        liquid in the vial has dropped on the bottom. Open the solvent        vial and put it in a vertical position.    -   2. To collect a sample from the vagina use the sterile flocked        swab provided with the TTD kit. Remove the sterile flocked swab        from its package following instructions on the package. The swab        tip should not touch anything prior to its insertion into        vagina. Hold the swab in the middle of the stick and, while the        patient is lying on her back, carefully insert the swab tip of        the swab into the vagina until the fingers contact the skin no        more than 2-3 inches (5-7 cm) deep. Withdraw the swab from the        vagina after 30 seconds.    -   3. Place the swab tip into the vial and rinse the swab in the        solvent by rotating for 30 seconds. Remove and dispose of the        swab.    -   4. Tear open the foil pouch at the tear notches and remove the        PAMG-1 test strip.    -   5. Dip the white end of the test strip (marked with arrows) into        the vial with solvent for no more than 5 minutes.    -   6. Remove the test strip if two stripes are clearly visible in        the vial (no later than 5 minutes sharp). Read the results by        placing the test on a clean, dry, flat surface.    -   7. Do not read or interpret the results after 10 minutes have        passed since dipping the test strip into the vial.    -   8. The presence of two lines indicates a positive test result        (positive for PAMG-1) and the presence of one line indicates a        negative test result (negative for PAMG-1).

Example 2: Clinical Trial for Predicting Time to Delivery (TTD)

A prospective observational clinical trial using the PAMG-1 detectionkit described in Example 1, above (referred to below as a TTD Test Kit),was run in order to assess the efficacy of the TTD test kit forpredicting TTD, based on the detection of PAMG-1 in the cervico-vaginalsecretions of pregnant women between 20^(0/7) and 36^(6/7) weeksgestational age presenting with signs and symptoms of PTL and havingclinically intact membranes.

Study Design:

The following study design is used:

1. Assessments are stratified by the following gestational age ranges:

-   -   a. <22 weeks    -   b. 22-34^(6/7) weeks    -   c. 35-36^(6/7) weeks

The TTD kit for the detection of PAMG-1 is compared to other methodsavailable in assessing time to delivery in the same patient population,including:

-   -   a. cervical length measurements by trans-vaginal ultrasound (<30        mm)    -   b. cervical dilatation>1 cm    -   c. contraction Frequency≥8 per hour

2. Data analysis

The association between the results of the TTD test, cervical length,and the following outcomes are determined:

-   -   a. Delivery<37 weeks gestation    -   b. Admission to neonatal intensive care unit (NICU)    -   c. Histological chorioamnionitis    -   d. Funisitis    -   e. Respiratory distress syndrome    -   f. Patent ductus arteriosus    -   g. Neonatal sepsis    -   h. Birth weight    -   i. Perinatal death

3. Selection and withdrawal of subjects:

The following includes and exclusion criteria was used:

-   -   Inclusion Criteria: Women between 20^(0/7) and 36^(6/7) weeks of        gestation with ≤3 cm cervical dilatation, presenting with        self-reported signs, symptoms or complaints suggestive of        preterm labor (outlined below) are invited to participate in the        trial:        -   a. Uterine contractions, with or without pain        -   b. Intermittent lower abdominal pain        -   c. Dull backache        -   d. Pelvic pressure        -   e. Bleeding during the second or third trimester        -   f. Menstrual-like or intestinal cramping, with or without            diarrhea        -   g. Absence of leakage from the cervical os observed via a            sterile speculum examination    -   Exclusion Criteria: During the clinical examination at        enrollment, subjects who are found to have one of the following        are deemed ineligible and are not included in the analysis:        -   a. Presented for regularly scheduled obstetrical care with            complaints of symptoms (i.e., the symptoms were not strong            enough in the patient's opinion to warrant unscheduled            emergency evaluation of her condition, such as would be            provided in a hospital Labor and Delivery Unit or Emergency            Room)        -   b. Received tocolytic medications for treatment of            threatened preterm delivery prior to collection of the            cervicovaginal specimens or cervical length measurements        -   c. Cervical dilatation>3 centimeters        -   d. Suspected placenta previa        -   e. <20^(0/7) weeks of gestation or ≥37 weeks of gestation        -   f. Overt rupture of the fetal membranes (ROM) as indicated            by visualized leakage of fluid from the cervical os        -   g. Cervical cerclage in place        -   h. A symptom not associated with idiopathic threatened            preterm delivery (e.g. trauma)        -   i. Digital exam prior to specimen collection        -   j. Enrollment in a tocolytic study        -   k. Heavy vaginal bleeding        -   l. <18 yrs old and not emancipated consenting minor

All patients undergoing labor augmentation to enhance the progression oflabor or who have a cesarean section delivery before active labor isdiagnosed (defined as regular contractions every 10 minutes or less,lasting more than 40 seconds, with cervical effacement more than 80percent and dilation of 2 cm (or 3 cm)) are not included in theanalysis.

4. Endpoints

Sensitivity (SN), specificity (SP), positive predictive value (PPV), andnegative predictive value (NPV) for the TTD test, cervical lengthmeasurements by trans-vaginal ultrasound (<30 mm), cervical dilatation>1cm, and contraction frequency≥8 per hour for the followingpresentation-to-delivery time intervals are determined:

-   -   a. ≤48 hours    -   b. ≤7 days    -   c. ≤14 days

5. Study Procedure

The following study procedure is followed:

-   -   a. Patients presenting with signs and symptoms of PTL who report        no intercourse within past 24 hours and were between 20^(0/7)        and 36^(6/7) weeks of gestation sign informed consent.    -   b. Specimen for the TTD test is collected (as described in        Example 1, above) prior to the insertion of a sterile speculum        examination in accordance with manufacturer's recommendations.    -   c. The sample is appropriately labeled and stored in a specially        designated place in accordance with manufacturer recommendations        for later examination by a separate investigator who was blinded        to the results of the physician's regular clinical evaluation.    -   d. After collecting the above-indicated sample, the physician        completes the physical examination of the patient to determine        whether the patient would be included or excluded from the        clinical trial based on the inclusion and exclusion criteria set        forth above.    -   e. Physician records the findings.    -   f. Cervical length measurement by transvaginal ultrasound (TVU)        is performed and results are recorded.    -   g. Patient delivery data (e.g. time, condition, etc.) are        recorded.    -   h. The collected sample is tested using the TTD test described        in Example 1.

6. Statistical Analysis

Mann-Whitney U test, Kaplan-Meier survival analysis and Cox regressionare used for evaluation of the primary outcome. PPV, NPV, SN, and SP arecalculated for the TTD test (for all time points tested). 95% confidenceintervals (CI) are computed using the Clopper-Pearson procedure.

7. Site Diagnosis and Management of Confirmed Preterm Labor Patient

In accordance with the ACOG Practice Bulletin on the Management ofPreterm Labor (2003), the patient diagnosed with PTL may be treated withone or more of the below:

-   -   a. Tocolytic therapy    -   b. Antibiotics    -   c. Bed Rest    -   d. Corticosteroids        Of the above possible combinations of diagnostic and treatment        options listed above, there is no supporting evidence indicating        that any have an effect on the primary outcome measure of this        study (which is the presentation-to-delivery time interval).

Results:

The expected results (based on present data) of an ongoing clinicaltrial are summarized in Table 3, below:

TABLE 2 Expected Results of Clinical Trial TTD NPV (%) PPV (%) SN (%) SP(%) (days) (95% CI*) (95% CI*) (95% CI*) (95% CI*) ≤2 100.0  45.5 100.0 86.7 (0.954, 1.00)  (0.244, 0.678) (0.692, 1.00)  (0.779, 0.929) ≤7 97.481.8 90.0 95.0 (0.910, 0.997) (0.597, 0.948) (0.683, 0.988) (0.877,0.986) ≤14 93.6 90.9 80.0 97.3 (0.857, 0.979) (0.708, 0.988) (0.593,0.932) (0.907, 0.997) Table Legend: “TTD”: time-to-delivery”; “SN”:sensitivity; “SP”: specificity; “NPV”: negative predictive value; “PPV”:positive predictive value; *95% confidence intervals (CI) computed viathe Clopper-Pearson procedure.

Final Results of Completed Clinical Trial

In the clinical trial, 101 women with singleton pregnancies between20^(0/7) and 36^(6/7) week of gestation presenting with self-reportedsigns, symptoms or complaints suggestive of preterm labor includinguterine contractions, with or without pain, intermittent lower abdominalpain and pelvic pressure were evaluated over the course of the study.The recruited patients had clinically intact amniotic membranes asdetermined by speculum examination and minimal cervical dilatation (≤3cm). A full clinical examination was conducted by the attendingphysician, including collection of the TTD test sample as described inExample 1, above. Parameters recorded at presentation included cervicallength, cervical dilatation, contraction frequency, membrane status,cervical effacement, patient history, and TTD Test Kit test result.Patients with overt rupture of fetal membranes, diagnosed as fluid seenleaking from the cervical os during the sterile speculum examination,were not enrolled in the study. The median age was 28 years (range:18-43 years), and the median gestational age at presentation was 31.4weeks (range: 22.4-36.5 weeks). No multiple gestations were included inthe trial.

The TTD test sample was collected and the TTD test procedure wasperformed as described in Example 1, above. The result was interpretedonce two lines were visible, or after five minutes elapsed since theinsertion of the test strip into the sample vial. The results werereported by the presence of two lines (positive for PAMG-1) or one line(negative for PAMG-1). The attending physician was not aware of the TTDtest results when making decisions about the care of the patient.Sensitivity (SN), specificity (SP), positive predictive value (PPV), andnegative predictive value (NPV) of the TTD test kit in predicting timeto spontaneous preterm delivery (within 7 and 14 days) were calculatedat the conclusion of the trial. 95% confidence intervals were calculatedusing the Clopper-Pearson procedure.

Twenty (20) patients delivered within 7 days of presentation and anadditional five delivered within 14 days of presentation. The TTD Testwas positive in 23% (23/101) of patients and the median test-to-deliveryinterval in this population was 3.86 days compared to 32.12 days for theTTD test negative group. Table 3, below, summarizes the TTD test resultsfor delivery within 7 and 14 days, respectively.

TABLE 3 Results of TTD Test Delivery ≤ 7 days Delivery ≤ 14 days (no.patients) (no. patients) + − + − TTD Test + 18 5 20 3 Results − 2 76 573

The NPV, PPV, SN and SP for the TTD Test confirming spontaneous pretermdelivery within 7 and 14 days of presentation are summarized in Table 4,below:

TABLE 4 NPV, PPV, SN and SP of TTD Test TTD NPV PPV SN SP (days) (95%CI*) (95% CI*) (95% CI*) (95% CI*) ≤7 97.4% 78.3% 90.0% 93.8%(91.0-99.7%) (56.3-92.5%) (68.3-98.8%) (86.2-98.0%) ≤14 93.6% 87.0%80.0% 96.1% (85.7-97.9%) (66.4-97.2%) (59.3-93.2%) (88.9-99.2%) TableLegend: “TTD”: time-to-delivery”; “SN”: sensitivity; “SP”: specificity;“NPV”: negative predictive value; “PPV”: positive predictive value; *95%confidence intervals (CI) computed via the Clopper-Pearson procedure.

The final NPV, PPV, SN and SP results for the TTD test for deliverywithin 48 hours (≤2 days) were the same as the expected results setforth in Table 2, above.

The incidence of a positive TTD test was also broken down by differentgestational age week ranges. The results are summarized in Table 5,below:

TABLE 5 Positive TTD Test Incidence and Performance by Gestational AgeGestational TTD Test Age Week Positive Delivery ≤ 7 days Interval(inclusive) Total Incidence SN SP PPV NPV 22-25  6  0.0% N/A 100.0% N/A100.0% 26-29 31 25.8% 100%  92.0%  75.0% 100.0% 30-33 37 24.3% 100% 90.3%  66.7% 100.0% 34-36 27 22.2%  75% 100.0% 100.0%  90.5% TableLegend: “TTD”: time-to-delivery; “SN”: sensitivity; “SP”: specificity;“NPV”: negative predictive value; “PPV”: positive predictive value

This Example, including the data shown in Tables 2-5, demonstrated thatthe TTD test provides a method for diagnosing TTD within 2, 7 or 14days, with high SN, SP, NPV, and PPV. It was demonstrated that the TTDtest can be used to rule out spontaneous preterm delivery within 2, 7and 14 days in patients with threatened preterm labor. A positive TTDtest in patients presenting with symptoms of preterm labor, intactmembranes, and minimal cervical dilatation (≤3 cm) indicated spontaneouspreterm delivery would occur within 7 days with a high degree ofaccuracy. A negative result, furthermore, indicated that spontaneouspreterm delivery within 14 days was highly unlikely. The Examples alsodemonstrates that the TTD test has a high PPV, NPV, SN and SP.

A number of embodiments of the present disclosure have been described.Nevertheless, it will be understood that various modifications may bemade without departing from the spirit and scope of the methodsdisclosed herein. It is further to be understood that all values areapproximate, and are provided for description. Accordingly, otherembodiments are within the scope of the following claims.

1.-2. (canceled)
 3. A method for determining a pregnant woman's risk ofspontaneous rupture of the chorioamniotic membranes, the methodcomprising: (a) contacting a vaginal fluid sample obtained from apregnant woman with at least two PAMG-1-specific monoclonal antibodies,wherein at least one of the antibodies binds to PAMG-1 when present inthe sample to form a PAMG-1/monoclonal antibody complex; (b) detectingthe presence of the PAMG-1/monoclonal antibody complex in the sampleonly when the concentration of PAMG-1 in the sample exceeds a predefineddetection threshold wherein the predetermined detection threshold levelof PAMG-1 is 4 ng/ml; and (c) determining that the pregnant woman is atrisk of spontaneous rupture of the chorioamniotic membranes if PAMG-1 isdetected; or (d) determining that the pregnant woman is not at risk ofspontaneous rupture of the chorioamniotic membranes if PAMG-1 is notdetected.
 4. The method of claim 3, further comprising determining thatthe fetal membranes of the pregnant woman are intact.
 5. The method ofclaim 3, comprising selecting the pregnant woman for analysis by themethod only if the pregnant woman presents with one or more of thefollowing: (i) signs, symptoms or complaints suggestive of pretermlabor; (ii) a gestational age between 20 weeks and 36 weeks, 6 days;(iii) a cervical length of 25 mm or more; and (iv) a cervical dilatationof 3 cm or less.
 6. The method of claim 5, comprising selecting thepregnant woman for analysis by the method only if the pregnant womanpresents with two or more of (i), (ii), (iii), and (iv).
 7. The methodof claim 6, comprising selecting the pregnant woman for analysis by themethod only if the pregnant woman presents with three or all four ofsigns (i), (ii), (iii), and (iv).
 8. The method of claim 3, comprisingcollecting the vaginal fluid sample from the pregnant woman with acollection device.
 9. The method of claim 8, wherein the collectiondevice is a flocked swab.
 10. The method of claim 9, wherein the flockedswab provides a 1:4 dilution of any PAMG-1 present in the vaginal fluidsample.
 11. The method of claim 8, comprising contacting the collectiondevice with a solvent to release the collected vaginal fluid sample. 12.The method of claim 3, comprising collecting the vaginal fluid sampleover a time period of about 30 seconds.
 13. The method of claim 11,comprising contacting the collection device with the solvent for about30 seconds after collecting the vaginal fluid sample.
 14. The method ofclaim 3, wherein the vaginal fluid sample is contacted with the at leasttwo PAMG-1-specific monoclonal antibodies for 5 minutes. 15.-20.(canceled)
 21. The method of claim 3, wherein the at least two PAMG-1specific monoclonal antibodies are used in a lateral flow device. 22.The method of claim 21, wherein the lateral flow device comprises a padregion and a test region.
 23. The method of claim 22, wherein the padregion of the test device comprises one of the at least two PAMG-1specific monoclonal antibodies and the test region comprises the otherof the two, and wherein the PAMG-1 specific monoclonal antibody in thepad region is mobilizable and the PAMG-1 specific monoclonal antibody inthe test region is immobilized.
 24. The method of claim 23, wherein thetest region of the test device further comprises a control region. 25.The method of claim 3, wherein one or more of the at least twoPAMG-1-specific monoclonal antibodies is an antibody selected from thegroup consisting of M271, produced by hybridoma N271, deposited with theRussian National Collection of Industrial Microorganisms (VKPM)Depository and assigned accession number VKPM-93; and M52, produced byhybridoma N52, deposited with the VKPM and assigned accession numberVKPM-92; and M42, produced by hybridoma N42, deposited with the VKPM andassigned accession number VKPM-94.
 26. The method of claim 25, where themobilizable antibody in the pad region is M271, produced by hybridomaN271, deposited with the Russian National Collection of IndustrialMicroorganisms (VKPM) Depository and assigned accession number VKPM-93,and the immobilized antibody in the test region is M52, produced byhybridoma N52, deposited with the VKPM and assigned accession numberVKPM-92. 27.-44. (canceled)
 45. The method of claim 9, wherein theflocked swab provides a dilution of any PAMG-1 present in the vaginalfluid sample in a range of 1:1 to 1:10.